Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma

ABSTRACT

Disclosed herein are therapeutic treatment protocols designed for the treatment of B cell lymphoma. These protocols are based upon therapeutic strategies which include the use of administration of immunologically active mouse/human chimeric anti-CD20 antibodies, radiolabeled anti-CD20 antibodies, and cooperative strategies comprising the use of chimeric anti-CD20 antibodies and radiolabeled anti-CD20 antibodies.

This application is a divisional, of application Ser. No. 08/149,099,pending, filed Nov. 3, 1993, which is a continuation-in-part of U.S.Ser. No. 07/978,891, filed Nov. 13, 1992, now abandoned.

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A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner does notobject to the reproduction by anyone of the patent document or thepatent disclosure, as it appears in the Patent and Trademark Officepatent files or records, but otherwise reserves all copyright rightswhatsoever.

RELATED APPLICATIONS

This patent document is related to U.S. Ser. No. 07/977,691, nowabandoned, entitled "IMPAIRED DOMINANT SELECTABLE MARKER SEQUENCE FORENHANCEMENT OF EXPRESSION OF CO-LINKED GENE PRODUCT AND EXPRESSIONVECTOR SYSTEMS COMPRISING SAME" having U.S. Ser. No. 07/977,691 (nowabandoned; filed Nov. 13, 1992) and "IMPAIRED DOMINANT SELECTABLE MARKERSEQUENCE AND INTRONIC INSERTION STRATEGIES FOR ENHANCEMENT OF EXPRESSIONOF GENE PRODUCT AND EXPRESSION VECTOR SYSTEMS COMPRISING SAME," U.S.Ser. No. 08/147,696, now U.S. Pat. No. 5,648,267 (filed simultaneouslyherewith). The related patent documents are incorporated herein byreference.

FIELD OF THE INVENTION

The references to be discussed throughout this document are set forthmerely for the information described therein prior to the filing datesof this document, and nothing herein is to be construed as an admission,either express or implied, that the references are "prior art" or thatthe inventors are not entitled to antedate such descriptions by virtueof prior inventions or priority based on earlier filed applications.

The present invention is directed to the treatment of B cell lymphomausing chimeric and radiolabeled antibodies to the B cell surface antigenBp35 ("CD20").

BACKGROUND OF THE INVENTION

The immune system of vertebrates (for example, primates, which includehumans, apes, monkeys, etc.) consists of a number of organs and celltypes which have evolved to: accurately and specifically recognizeforeign microorganisms ("antigen") which invade the vertebrate-host;specifically bind to such foreign microorganisms; and, eliminate/destroysuch foreign microorganisms. Lymphocytes, amongst others, are criticalto the immune system. Lymphocytes are produced in the thymus, spleen andbone marrow (adult) and represent about 30% of the total white bloodcells present in the circulatory system of humans (adult). There are twomajor sub-populations of lymphocytes: T cells and B cells. T cells areresponsible for cell mediated immunity, while B cells are responsiblefor antibody production (humoral immunity). However, T cells and B cellscan be considered as interdependent--in a typical immune response, Tcells are activated when the T cell receptor binds to fragments of anantigen that are bound to major histocompatability complex ("MHC")glycoproteins on the surface of an antigen presenting cell; suchactivation causes release of biological mediators ("interleukins")which, in essence, stimulate B cells to differentiate and produceantibody ("immunoglobulins") against the antigen.

Each B cell within the host expresses A different antibody on itssurface-thus, one B cell will express antibody specific for one antigen,while another B cell will express antibody specific for a differentantigen. Accordingly, B cells are quite diverse, and this diversity iscritical to the immune system. In humans, each B cell can produce anenormous number of antibody molecules (ie, about 10⁷ to 10⁸). Suchantibody production most typically ceases (or substantially decreases)when the foreign antigen has been neutralized. Occasionally, however,proliferation of a particular B cell will continue unabated; suchproliferation can result in a cancer referred to as "B cell lymphoma."

T cells and B cells both comprise cell surface proteins which can beutilized as "markers" for differentiation and identification. One suchhuman B cell marker is the human B lymphocyte-restricted differentiationantigen Bp35, referred to as "CD20." CD20 is expressed during earlypre-B cell development and remains until plasma cell differentiation.Specifically, the CD20 molecule may regulate a step in the activationprocess which is required for cell cycle initiation and differentiationand is usually expressed at very high levels on neoplastic ("tumor") Bcells. CD20, by definition, is present on both "normal" B cells as wellas "malignant" B cells, ie, those B cells whose unabated proliferationcan lead to B cell lymphoma. Thus, the CD20 surface antigen has thepotential of serving as a candidate for "targeting" of B cell lymphomas.

In essence, such targeting can be generalized as follows: antibodiesspecific to the CD20 surface antigen of B cells are, eg, injected into apatient. These anti-CD20 antibodies specifically bind to the CD20 cellsurface antigen of (ostensibly) both normal and malignant B cells; theanti-CD20 antibody bound to the CD20 surface antigen may lead to thedestruction and depletion of neoplastic B cells. Additionally, chemicalagents or radioactive labels having the potential to destroy the tumorcan be conjugated to the anti-CD20 antibody such that the agent isspecifically "delivered " to, eg, the neoplastic B cells. Irrespectiveof the approach, a primary goal is to destroy the tumor: the specificapproach can be determined by the particular anti-CD20 antibody which isutilized and, thus, the available approaches to targeting the CD20antigen can vary considerably.

For example, attempts at such targeting of CD20 surface antigen havebeen reported. Murine (mouse) monoclonal antibody 1F5 (an anti-CD20antibody) was reportedly administered by continuous intravenous infusionto B cell lymphoma patients. Extremely high levels (>2 grams) of 1F5were reportedly required to deplete circulating tumor cells, and theresults were described as being "transient." Press et al., "MonoclonalAntibody 1F5 (Anti-CD20) Serotherapy of Human B-Cell Lymphomas." Blood69/2:584-591 (1987). A potential problem with this approach is thatnon-human monoclonal antibodies (eg, murine monoclonal antibodies)typically lack human effector functionality, eg, they are unable to,inter alia, mediate complement dependent lysis or lyse human targetcells through antibody dependent cellular toxicity or Fc-receptormediated phagocytosis. Furthermore, non-human monoclonal antibodies canbe recognized by the human host as a foreign protein; therefore,repeated injections of such foreign antibodies can lead to the inductionof immune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody response, or "HAMA" response. Additionally, these"foreign" antibodies can be attacked by the immune system of the hostsuch that they are, in effect, neutralized before they reach theirtarget site.

Lymphocytes and lymphoma cells are inherently sensitive to radiotherapyfor several reasons: the local emission of ionizing radiation ofradiolabeled antibodies may kill cells with or without the targetantigen (eg, CD20) in close proximity to antibody bound to the antigen;penetrating radiation may obviate the problem of limited access to theantibody in bulky or poorly vascularized tumors; and, the total amountof antibody required may be reduced. The radionuclide emits radioactiveparticles which can damage cellular DNA to the point where the cellularrepair mechanisms are unable to allow the cell to continue living;therefore, if the target cells are tumors, the radioactive labelbeneficially kills the tumor cells. Radiolabeled antibodies, bydefinition, include the use of a radioactive substance which may requirethe need for precautions for both the patient (ie, possible bone marrowtransplantation) as well as the health care provider (ie, the need toexercise a high degree of caution when working with the radioactivity).

Therefore, an approach at improving the ability of murine monoclonalantibodies to be effective in the treatment of B-cell disorders has beento conjugate a radioactive label or toxin to the antibody such that thelabel or toxin is localized at the tumor site. For example, theabove-referenced IF5 antibody has been "labeled" with iodine-131 ("¹³¹I") and was reportedly evaluated for biodistribution in two patients.See Eary, J. F. et al., "Imaging and Treatment of B-Cell Lymphoma" J.Nuc. Med. 31/8:1257-1268 (1990); see also, Press, O. W. et al.,"Treatment of Refractory Non-Hodgkin's Lymphoma with Radiolabeled MB-1(Anti-CD37) Antibody" J. Clin. Onc. 718:1027-1038 (1989) (indicationthat one patient treated with ¹³¹ I-labeled IF-5 achieved a "partialresponse"); Goldenberg, D. M. et al., "Targeting, Dosimetry andRadioimmunotherapy of B-Cell Lymphomas with Iodine-131-Labeled LL2Monoclonal Antibody" J. Clin. Onc. 9/4:548-564 (1991) (three of eightpatients receiving multiple injections reported to have developed a HAMAresponse); Appelbaum, F. R. "Radiolabeled Monoclonal Antibodies in theTreatment of Non-Hodgkin's Lymphoma" Hem./Onc. Clinics of N. A.5/5:1013-1025 (1991) (review article); Press, O. W. et al"Radiolabeled-Antibody Therapy of B-Cell Lymphoma with Autologous BoneMarrow Support." New England Journal of Medicine 329/17: 1219-12223(1993) (iodine-131 labeled anti-CD20 antibody IF5 and B1); and Kaminski,M. G. et al "Radioimmunotherapy of B-Cell Lymphoma with ¹³¹ I! Anti-B1(Anti-CD20) Antibody". NEJM 329/7 (1993) (iodine-131 labeled anti-CD20antibody B1; hereinafter "Kaminski").

Toxins (ie chemotherapeutic agents such as doxorubicin or mitomycin C)have also been conjugated to antibodies. See, for example, PCT publishedapplication WO 92/07466 (published May 14, 1992).

"Chimeric" antibodies, ie, antibodies which comprise portions from twoor more different species (eg, mouse and human) have been developed asan alternative to "conjugated" antibodies. For example, Liu, A. Y. etal., "Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20with Potent Fc-Dependent Biologic Activity" J. Immun. 139 /10:3521-3526(1987), describes a mouse/human chimeric antibody directed against theCD20 antigen. See also, PCT Publication No. WO 88/04936. However, noinformation is provided as to the ability, efficacy or practicality ofusing such chimeric antibodies for the treatment of B cell disorders inthe reference. It is noted that in vitro functional assays (eg,complement dependent lysis ("CDC"); antibody dependent cellularcytotoxicity ("ADCC"), etc.) cannot inherently predict the in vivocapability of a chimeric antibody to destroy or deplete target cellsexpressing the specific antigen. See, for example, Robinson, R. D. etal., "Chimeric mouse-human anti-carcinoma antibodies that mediatedifferent anti-tumor cell biological activities," Hum. Antibod.Hybridomas 2:84-93 (1991) (chimeric mouse-human antibody havingundetectable ADCC activity). Therefore, the potential therapeuticefficacy of chimeric antibody can only truly be assessed by in vivoexperimentation.

What is needed, and what would be a great advance in the art, aretherapeutic approaches targeting the CD20 antigen for the treatment of Bcell lymphomas in primates, including, but not limited to, humans.

SUMMARY OF THE INVENTION

Disclosed herein are therapeutic methods designed for the treatment of Bcell disorders, and in particular, B cell lymphomas. These protocols arebased upon the administration of immunologically active chimericanti-CD20 antibodies for the depletion of peripheral blood B cells,including B cells associated with lymphoma; administration ofradiolabeled anti-CD20 antibodies for targeting localized and peripheralB cell associated tumors; and administration of chimeric anti-CD20antibodies and radiolabeled anti-CD20 antibodies in a cooperativetherapeutic strategy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of a tandem chimeric antibodyexpression vector useful in the production of immunologically activechimeric anti-CD20 antibodies ("TCAE 8");

FIGS. 2A through 2F are the nucleic acid sequence of the vector of FIG.1;

FIGS. 3A through 3F are the nucleic acid sequence of the vector of FIG.1 further comprising murine light and heavy chain variable regions("anti-CD20 in TCAE 8");

FIG. 4 is the nucleic acid and amino acid sequences (including CDR andframework regions) of murine variable region light chain derived frommurine anti-CD20 monoclonal antibody 2B8;

FIG. 5 is the nucleic acid and amino acid sequences (including CDR andframework regions) of murine variable region heavy chain derived frommurine anti-CD20 monoclonal antibody 2B8;

FIG. 6 are flow cytometry results evidencing binding offluorescent-labeled human C1q to chimeric anti-CD20 antibody, including,as controls labeled C1q; labeled C1q and murine anti-CD20 monoclonalantibody 2B8; and labeled C1q and human IgGl,k;

FIG. 7 represents the results of complement related lysis comparingchimeric anti-CD20 antibody and murine anti-CD20 monoclonal antibody2B8;

FIG. 8 represents the results of antibody mediated cellular cytotoxicitywith in vivo human effector cells comparing chimeric anti-CD20 antibodyand 2B8;

FIG. 9A, 9B and 9C provide the results of non-human primate peripheralblood B lymphocyte depletion after infusion of 0.4 mg/kg (A); 1.6 mg/kg(B); and 6.4 mg/kg (C) of immunologically active chimeric anti-CD20antibody;

FIG. 10 provides the results of, inter alia, non-human primateperipheral blood B lymphocyte depletion after infusion of 0.01 mg/kg ofimmunologically active chimeric anti-CD20 antibody;

FIG. 11 provides results of the tumoricidal impact of Y2B8 in a mousexenographic model utilizing a B cell lymphoblastic tumor;

FIG. 12 provides results of the tumoricidal impact of C2B8 in a mousexenographic model utilizing a B cell lymphoblastic tumor;

FIG. 13 provides results of the tumoricidal impact of a combination ofY2B8 and C2B8 in a mouse xenographic model utilizing a B celllymphoblastic tumor; and

FIGS. 14A and 14B provide results from a Phase I/II clinical analysis ofC2B8 evidencing B-cell population depletion over time for patientsevidencing a partial remission of the disease (14A) and a minorremission of the disease (14B).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Generally, antibodies are composed of two light chains and two heavychain molecules; these chains form a general "Y" shape, with both lightand heavy chains forming the arms of the Y and the heavy chains formingthe base of the Y. Light and heavy chains are divided into domains ofstructural and functional homology. The variable domains of both thelight ("V_(L) ") and the heavy ("V_(H) ") chains determine recognitionand specificity. The constant region domains of light ("C_(L) ") andheavy ("C_(H) ") chains confer important biological properties, egantibody chain association, secretion, transplacental mobility, Fcreceptor binding complement binding, etc. The series of events leadingto immunoglobulin gene expression in the antibody producing cells arecomplex. The variable domain region gene sequences are located inseparate germ line gene segments referred to as "V_(H)," "D," and"J_(H)," or "V_(L) " and "J_(L)." These gene segments are joined by DNArearrangements to form the complete V regions expressed in heavy andlight chains, respectively. The rearranged, joined V segments (V_(L)-J_(L) and V_(H) -D-J_(H)) then encode the complete variable regions orantigen binding domains of light and heavy chains, respectively.

Serotherapy of human B cell lymphomas using an anti-CD20 murinemonoclonal antibody (1F5) has been described by Press et al., (69 Blood584, 1987, supra); the reported therapeutic responses, unfortunately,were transient. Additionally, 25% of the tested patients reportedlydeveloped a human anti-mouse antibody (HAMA) response to theserotherapy. Press et al., suggest that these antibodies, conjugated totoxins or radioisotopes, might afford a more lasting clinical benefitthan the unconjugated antibody.

Owing to the debilitating effects of B cell lymphoma and the very realneed to provide viable treatment approaches to this disease, we haveembarked upon different approaches having a particular antibody, 2B8, asthe common link between the approaches. One such approach advantageouslyexploits the ability of mammalian systems to readily and efficientlyrecover peripheral blood B cells; using this approach, we seek to, inessence, purge or deplete B cells in peripheral blood and lymphatictissue as a means of also removing B cell lymphomas. We accomplish thisby utilization of, inter alia, immunologically active, chimericanti-CD20 antibodies. In another approach, we seek to target tumor cellsfor destruction with radioactive labels.

As used herein, the term "anti-CD20 antibody" is an antibody whichspecifically recognizes a cell surface non-glycosylated phosphoproteinof 35,000 Daltons, typically designated as the human B lymphocyterestricted differentiation antigen Bp35, commonly referred to as CD20.As used herein, the term "chimeric" when used in reference to anti-CD20antibodies, encompasses antibodies which are most preferably derivedusing recombinant deoxyribonucleic acid techniques and which compriseboth human (including immunologically "related" species, eg, chimpanzee)and non-human components: the constant region of the chimeric antibodyis most preferably substantially identical to the constant region of anatural human antibody; the variable region of the chimeric antibody ismost preferably derived from a non-human source and has the desiredantigenic and specificity to the CD20 cell surface antigen. Thenon-human source can be any vertebrate source which can be used togenerate antibodies to a human CD20 cell surface antigen or materialcomprising a human CD20 cell surface antigen. Such non-human sourceincludes, but is not limited to, rodents (eg, rabbit, rat, mouse, etc.)and non-human primates (eg, Old World Monkey, Ape, etc.). Mostpreferably, the non-human component (variable region) is derived from amurine source. As used herein, the phrase "immunologically active" whenused in reference to chimeric anti-CD20 antibodies, means a chimericantibody which binds human C1q, mediates complement dependent lysis("CDC") of human B lymphoid cell lines, and lyses human target cellsthrough antibody dependent cellular cytotoxicity ("ADCC"). As usedherein, the phrases "indirect labeling" and "indirect labeling approach"both mean that a chelating agent is covalently attached to an antibodyand at least one radionuclide is inserted into the chelating agent.Preferred chelating agents and radionuclides are set forth inSrivagtava, S. C. and Mease, R. C.,"Progress in Research on Ligands,Nuclides and Techniques for Labeling Monoclonal Antibodies," Nucl. Med.Bio. 18/6: 589-603 (1991) ("Srivagtava") which is incorporated herein byreference. A particularly preferred chelating agent is1-isothiocycmatobenzyl-3-methyldiothelene triaminepent acetic acid("MX-DTPA"); particularly preferred radionuclides for indirect labelinginclude indium 111! and yttrium 90!. As used herein, the phrases "directlabeling" and "direct labeling approach" both mean that a radionuclideis covalently attached directly to an antibody (typically via an aminoacid residue). Preferred radionuclides are provided in Srivagtava; aparticularly preferred radionuclide for direct labeling is iodine 131!covalently attached via tyrosine residues. The indirect labelingapproach is particularly preferred.

The therapeutic approaches disclosed herein are based upon the abilityof the immune system of primates to rapidly recover, or rejuvenate,peripheral blood B cells. Additionally, because the principal immuneresponse of primates is occasioned by T cells, when the immune systemhas a peripheral blood B cell deficiency, the need for "extraordinary"precautions (ie patient isolation, etc.) is not necessary. As a resultof these and other nuances of the immune systems of primates, ourtherapeutic approach to B cell disorders allows for the purging ofperipheral blood B cells using immunologically active chimeric anti-CD20antibodies.

Because peripheral blood B cell disorders, by definition, can indicate anecessity for access to the blood for treatment, the route ofadministration of the immunologically active chimeric anti-CD20antibodies and radioalabeled anti-CD20 antibodies is preferablyparenteral; as used herein, the term "parenteral" includes intravenous,intramuscular, subcutaneous, rectal, vaginal or intraperitonealadministration. Of these, intravenous administration is most preferred.

The immunologically active chimeric anti-CD20 antibodies andradiolabeled anti-CD20 antibodies will typically be provided by standardtechnique within a pharmaceutically acceptable buffer, for example,sterile saline, sterile buffered water, propylene glycol, combinationsof the foregoing, etc. Methods for preparing parenterally administrableagents are described in Pharmaceutical Carriers & Formulations, Martin,Remington's Pharmaceutical Sciences, 15th Ed. (Mack Pub. Co., Easton,Pa. 1975), which is incorporated herein by reference.

The specific, therapeutically effective amount of immunologically activechimeric anti-CD20 antibodies useful to produce a unique therapeuticeffect in any given patient can be determined by standard techniqueswell known to those of ordinary skill in the art.

Effective dosages (ie, therapeutically effective amounts) of theimmunologically active chimeric anti-CD20 antibodies range from about0.001 to about 30 mg/kg body weight, more preferably from about 0.01 toabout 25 mg/kg body weight, and most preferably from about 0.4 to about20.0 mg/kg body weight. Other dosages are viable; factors influencingdosage include, but are not limited to, the severity of the disease;previous treatment approaches; overall health of the patient; otherdiseases present, etc. The skilled artisan is readily credited withassessing a particular patient and determining a suitable dosage thatfalls within the ranges, or if necessary, outside of the ranges.

Introduction of the immunologically active chimeric anti-CD20 antibodiesin these dose ranges can be carried out as a single treatment or over aseries of treatments. With respect to chimeric antibodies, it ispreferred that such introduction be carried out over a series oftreatments; this preferred approach is predicated upon the treatmentmethodology associated with this disease. While not wishing to be boundby any particular theory, because the immunologically active chimericanti-CD20 antibodies are both immunologically active and bind to CD20,upon initial introduction of the immunologically active chimericanti-CD20 antibodies to the individual, peripheral blood B celldepletion will begin; we have observed a nearly complete depletionwithin about 24 hours post treatment infusion. Because of this,subsequent introduction(s) of the immunologically active chimericanti-CD20 antibodies (or radiolabeled anti-CD20 antibodies) to thepatient is presumed to: a) clear remaining peripheral blood B cells; b)begin B cell depletion from lymph nodes; c) begin B cell depletion fromother tissue sources, eg, bone marrow, tumor, etc. Stated again, byusing repeated introductions of the immunologically active chimericanti-CD20 antibodies, a series of events take place, each event beingviewed by us as important to effective treatment of the disease. Thefirst "event" then, can be viewed as principally directed tosubstantially depleting the patient's peripheral blood B cells; thesubsequent "events" can be viewed as either principally directed tosimultaneously or serially clearing remaining B cells from the systemclearing lymph node B cells, or clearing other tissue B cells.

In effect, while a single dosage provides benefits and can beeffectively utilized for disease treatment/management, a preferredtreatment course can occur over several stages; most preferably, betweenabout 0.4 and about 20 mg/kg body weight of the immunologically activechimeric anti-CD20 antibodies is introduced to the patient once a weekfor between about 2 to 10 weeks, most preferably for about 4 weeks.

With reference to the use of radiolabeled anti-CD20 antibodies, apreference is that the antibody is non-chimeric; this preference ispredicted upon the significantly longer circulating half-life ofchimeric antibodies vis-a-vis murine antibodies (ie, with a longercirculating half-life, the radionuclide is present in the patient forextended periods). However, radiolabeled chimeric antibodies can bebeneficially utilized with lower milli-Curries ("mCi") dosages used inconjunction with the chimeric antibody relative to the murine antibody.This scenario allows for a decrease in bone marrow toxicity to anacceptable level, while maintaining therapeutic utility.

A variety of radionuclides are applicable to the present invention andthose skilled in the art are credited with the ability to readilydetermine which radionuclide is most appropriate under a variety ofcircumstances. For example, iodine 131! is a well known radionuclideused for targeted immunotherapy. However, the clinical usefulness ofiodine 131! can be limited by several factors including: eight-dayphysical half-life; dehalogenation of iodinated antibody both in theblood and at tumor sites; and emission characteristics (eg, large gammacomponent) which can be suboptimal for localized dose deposition intumor. With the advent of superior chelating agents, the opportunity forattaching metal chelating groups to proteins has increased theopportunities to utilize other radionuclides such as indium 131! andyttrium 90!. Yttrium 90! provides several benefits for utilization inradioimmunotherapeutic applications: the 64 hour half-life of yttrium90! is long enough to allow antibody accumulation by tumor and, unlikeeg, iodine 131!, yttrium 90! is a pure beta emitter of high energy withno accompanying gamma irradiation in its decay, with a range in tissueof 100 to 1000 cell diameters. Furthermore, the minimal amount ofpenetrating radiation allows for outpatient administration of yttrium90!-labeled antibodies. Additionally, interalization of labeled antibodyis not required for cell killing, and the local emission of ionizingradiation should be lethal for adjacent tumor cells lacking the targetantigen.

One non-therapeutic limitation to yttrium 90! is based upon the absenceof significant gamma radiation making imaging therewith difficult. Toavoid this problem, a diagnostic "imaging" radionuclide, such as indium111!, can be utilized for determining the location and relative size ofa tumor prior to the administration of therapeutic does of yttrium90!-labeled anti-CD20. Indium 111! is particularly preferred as thediagnostic radionuclide because: between about 1 to about 10 mCi can besafely administered without detectable toxicity; and the imaging data isgenerally predictive of subsequent yttrium 90!-labeled antibodydistribution. Most imaging studies utilize 5 mCi indium 111!-labeledantibody because this dose is both safe and has increased imagingefficiency compared with lower doses, with optimal imaging occurring atthree to six days after antibody administration. See, for example,Murray J. L., 26 J. Nuc. Med. 3328 (1985) and Carraguillo, J. A. et al,26 J. Nuc. Med. 67 (1985).

Effective single treatment dosages (ie, therapeutically effectiveamounts) of yttrium 90! labeled anti-CD20 antibodies range from betweenabout 5 and about 75 mCi, more preferably between about 10 and about 40mCi. Effective single treatment non-marrow ablative dosages of iodine131! labeled anti-CD20 antibodies range from between about 5 and about70 mCi, more preferably between about 5 and about 40 mCi. Effectivesingle treatment ablative dosages (ie, may require autologous bonemarrow transplantation) of iodine 131! labeled anti-CD20 antibodiesrange from between about 30 and about 600 mCi, more preferably betweenabout 50 and less than about 500 mCi. In conjunction with a chimericanti-CD20 antibody, owing to the longer circulating half life vis-a-vismurine antibodies, an effective single treatment non-marrow ablativedosages of iodine 131! labeled chimeric anti-CD20 antibodies range frombetween about 5 and about 40 mCi, more preferably less than about 30mCi. Imaging criteria for, eg, the indium 111! label, are typically lessthan about 5 mCi.

With respect to radiolabeled anti-CD20 antibodies, therapy therewith canalso occur using a single therapy treatment or using multipletreatments. Because of the radionuclide component, it is preferred thatprior to treatment, peripheral stem cells ("PSC") or bone marrow ("BM")be "harvested" for patients experiencing potentially fatal bone marrowtoxicity resulting from radiation. BM and/or PSC are harvested usingstandard techniques, and then purged and frozen for possible reinfusion.Additionally, it is most preferred that prior to treatment a diagnosticdosimetry study using a diagnostic labeled antibody (eg, using indium111!) be conducted on the patient, a purpose of which is to ensure thatthe therapeutically labeled antibody (eg, using yttrium 90!) will notbecome unnecessarily "concentrated" in any normal organ or tissue.

Chimeric mouse/human antibodies have been described. See, for example,Morrison, S. L. et al., PNAS I1:6851-6854 (November 1984); EuropeanPatent Publication No. 172494; Boulianne, G. L. at al., Nature 312;642(December 1984); Neubeiger, M. S. et al., Nature 314:268 (March 1985);European Patent Publication No. 125023; Tan et al., J. Immunol. 135:8564(November 1985); Sun, L. K. et al., Hybridoma 5/1:517 (1986); Sahagan etal., J. Immunol. 137:1066-1074 (1986). See generally, Muron, Nature312:597 (December 1984); Dickson, Genetic Engineering News 5/3 (March1985); Marx, Science 229 455 (August 1985); and Morrison Science229:1202-1207 (September 1985). Robinson et al., in PCT PublicationNumber WO 88/04936, describe a chimeric antibody with human constantregion and murine variable region, having specificity to an epitope ofCD20; the murine portion of the chimeric antibody of the Robinsonreferences is derived from the 2H7 mouse monoclonal antibody (gamma 2b,kappa). While the reference notes that the described chimeric antibodyis a "prime candidate" for the treatment of B cell disorders, thisstatement can be viewed as no more than a suggestion to those in the artto determine whether or not this suggestion is accurate for thisparticular antibody, particularly because the reference lacks any datato support an assertion of therapeutic effectiveness, and importantly,data using higher order mammals such as primates or humans.

Methodologies for generating chimeric antibodies are available to thosein the art. For example, the light and heavy chains can be expressedseparately, using, for example, immunoglobulin light chain andimmunoglobulin heavy chains in separate plasmids. These can then bepurified and assembled in vitro into complete antibodies; methodologiesfor accomplishing such assembly have been described. See, for example,Scharff, M., Harvey Lectures 69:125 (1974). In vitro reaction parametersfor the formation of IgG antibodies from reduced isolated light andheavy chains have also been described. See, for example, Beychok, S.,Cells of Immunoglobulin Synthesis, Academic Press, New York, p. 69,1979. Co-expression of light and heavy chains in the same cells toachieve intracellular association and linkage of heavy and light chainsinto complete H₂ L₂ IgG antibodies is also possible. Such co-expressioncan be accomplished using either the same or different plasmids in thesame host cell.

Another approach, and one which is our most preferred approach fordeveloping a chimeric non-human/human anti-CD20 antibody, is based uponutilization of an expression vector which includes, ab initio, DNAencoding heavy and light chain constant regions from a human source.Such a vector allows for inserting DNA encoding non-human variableregion such that a variety of non-human anti-CD20 antibodies can begenerated, screened and analyzed for various characteristics (eg, typeof binding specificity, epitope binding regions, etc.); thereafter, cDNAencoding the light and heavy chain variable regions from a preferred ordesired anti-CD20 antibody can be incorporated into the vector. We referto these types of vectors as Tandem Chimeric Antibody Expression("TCAE") vectors. A most preferred TCAE vector which was used togenerate immunologically active chimeric anti-CD20 antibodies fortherapeutic treatment of lymphomas is TCAE 8. TCAE 8 is a derivative ofa vector owned by the assignee of this patent document, referred to asTCAE 5.2 the difference being that in TCAE 5.2, the translationinitiation start site of the dominant selectable marker (neomycinphosphostransferase, "NEO") is a consensus Kozak sequence, while forTCAE 8, this region is a partially impaired consensus Kozak sequence.Details regarding the impact of the initiation start site of thedominant selectable marker of the TCAE vectors (also referred to as"ANEX vector") vis-a-vis protein expression are disclosed in detail inthe co-pending application filed herewith. TCAE 8 comprises four (4)transcriptional cassettes, and these are in tandem order, ie, a humanimmunoglobulin light chain absent a variable region; a humanimmunoglobulin heavy chain absent a variable region; DHFR; and NEO. Eachtranscriptional cassette contains its own eukaryotic promoter andpolyadenylation region (reference is made to FIG. 1 which is adiagrammatic representation of the TCAE 8 vector). Specifically:

1) the CMV promoter/enhancer in front of the immunoglobulin heavy chainis a truncated version of the promoter/enhancer in front of the lightchain, from the Nhe I site at -350 to the Sst I site at -16 (see, 41Cell 521, 1985).

2) a human immunoglobulin light chain constant region was derived viaamplification of cDNA by a PCR reaction. In TCAE 8, this was the humanimmunoglobulin light chain kappa constant region (Kabat numbering, aminoacids 108-214, allotype Km 3, (see, Kabat, E. A. "Sequences of proteinsof immunological interest," NIH Publication, Fifth Ed. No. 91-3242,1991)), and the human immunoglobulin heavy chain gamma 1 constant region(Kabat numbering amino acids 114-478, allotype Gmla, Gmlz). The lightchain was isolated from normal human blood (IDEC PharmaceuticalsCorporation, La Jolla, Calif.); RNA therefrom was used to synthesizecDNA which was then amplified using PCR techniques (primers were derivedvis-a-vis the consensus from Kabat). The heavy chain was isolated (usingPCR techniques) from cDNA prepared from RNA which was in turn derivedfrom cells transfected with a human IgG1 vector (see, 3 Prot. Eng 531,1990; vector pN.sub.γ1 62). Two amino acids were changed in the isolatedhuman IgG1 to match the consensus amino acid sequence from Kabat, towit: amino acid 225 was changed from valine to alanine (GTT to GCA), andamino acid 287 was changed from methionine to lysine (ATG to AAG);

3) The human immunoglobulin light and heavy chain cassettes containsynthetic signal sequences for secretion of the immunoglobulin chains;

4) The human immunoglobulin light and heavy chain cassettes containspecific DNA restriction sites which allow for insertion of light andheavy immunoglobulin variable regions which maintain the transitionalreading frame and do not alter the amino acids normally found inimmunoglobulin chains;

5) The DHFR cassette contained its own eukaryotic promoter (mouse betaglobin major promoter, "BETA") and polyadenylation region (bovine growthhormone polyadenylation, "BGH"); and

6) The NEO cassette contained its own eukaryotic promoter (BETA) andpolyadenylation region (SV40 early polyadenylation, "SV").

With respect to the TCAE 8 vector and the NEO cassette, the Kozak regionwas a partially impaired consensus Kozak sequence (which included anupstream Cla I site): ##STR1## (In the TCAE 5.2 vector, the change isbetween the Cla I and ATG regions, to wit: ccAcc.)

The complete sequence listing of TCAE 8 (including the specificcomponents of the four transcriptional cassettes) is set forth in FIG. 2(SEQ. ID. NO. 1).

As will be appreciated by those in the art, the TCAE vectorsbeneficially allow for substantially reducing the time in generating theimmunologically active chimeric anti-CD20 antibodies. Generation andisolation of non-human light and heavy chain variable regions, followedby incorporation thereof within the human light chain constanttranscriptional cassette and human heavy chain constant transcriptionalcassette, allows for production of immunologically active chimericanti-CD20 antibodies.

We have derived a most preferred non-human variable region withspecificity to the CD20 antigen using a murine source and hybridomatechnology. Using polymerase chain reaction ("PCR") techniques, themurine light and heavy variable regions were cloned directly into theTCAE 8 vector--this is the most preferred route for incorporation of thenon-human variable region into the TCAE vector. This preference isprincipally predicated upon the efficiency of the PCR reaction and theaccuracy of insertion. However, other equivalent procedures foraccomplishing this task are available. For example, using TCAE 8 (or anequivalent vector), the sequence of the variable region of a non-humananti-CD20 antibody can be obtained, followed by oligonucleotidesynthesis of portions of the sequence or, if appropriate, the entiresequence; thereafter, the portions or the entire synthetic sequence canbe inserted into the appropriate locations within the vector. Thoseskilled in the art are credited with the ability to accomplish thistask.

Our most preferred immunologically active chimeric anti-CD20 antibodieswere derived from utilization of TCAE 8 vector which included murinevariable regions derived from monoclonal antibody to CD20; this antibody(to be discussed in detail, infra), is referred to as "2B8." Thecomplete sequence of the variable regions obtained from 2B8 in TCAE 8("anti-CD20 in TCAE 8") is set forth in FIG. 3 (SEQ. ID. NO. 2).

The host cell line utilized for protein expression is most preferably ofmammalian origin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein, Exemplaryhost cell lines include, but are not limited to, DG44 and DUXBll(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervicalcarcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mousefibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma),P3×63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovine endothelial cells),RAJI (human lymphocyte) and 293 (human kidney). Host cell lines aretypically available from commercial services, the American TissueCulture Collection or from published literature.

Preferably the host cell line is either DG44 ("CHO") or SP2/0. SeeUrland, G. et al., "Effect of gamma rays and the dihydrofolate reductaselocus: deletions and inversions." Som. Cell & Mol. Gen. 12/6:555-566(1986), and Shulman, M. et al., "A better cell line for makinghybridomas secreting specific antibodies." Nature 276:269 (1978),respectively. Most preferably, the host cell line is DG44. Transfectionof the plasmid into the host cell can be accomplished by any techniqueavailable to those in the art. These include, but are not limited to,transfection (including electrophoresis and electroporation), cellfusion with enveloped DNA, microinjection, and infection with intactvirus. See, Ridgway, A. A. G. "Mammalian Expression Vectors." Chapter24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths,Boston, Mass. 1988). Most preferably, plasmid introduction into the hostis via electroporation.

EXAMPLES

The following examples are not intended, nor are they to be construed,as limiting the invention. The examples are intended to evidence;dose-imaging using a radiolabeled anti-CD20 antibody ("I2B8");radiolabeled anti-CD20 antibody ("Y2B8"); and immunologically active,chimeric anti-CD20 antibody ("C2B8") derived utilizing a specific vector("TCAE 8") and variable regions derived from murine anti-CD20 monoclonalantibody ("2B8").

I. RADIOLABELED ANTI-CD20 ANTIBODY 2B8

A. Anti-CD20 Monoclonal Antibody (Murine) Production ("2B8")

BALB/C mice were repeatedly immunized with the human lymphoblastoid cellline SB (see, Adams, R. A. et al., "Direct implantation and serialtransplantation of human acute lymphoblastic leukemia in hamsters,SB-2." Can Res 28:1121-1125 (1968); this cell line is available from theAmerican Tissue Culture Collection, Rockville, Md., under ATCC accessionnumber ATCC CCL 120), with weekly injections over a period of 3-4months. Mice evidencing high serum titers of anti-CD20 antibodies, asdetermined by inhibition of known CD20-specific antibodies (anti-CD20antibodies utilized were Leu 16, Beckton Dickinson, San Jose, Calif,Cat. No. 7670; and Bl, Coulter Corp., Hialeah, Fla., Cat. No. 6602201)were identified; the spleens of such mice were then removed. Spleencells were fused with the mouse myeloma SP2/0 in accordance with theprotocol described in Einfeld, D. A. et al., (1988) EMBO 7:711 (SP2/0has ATCC accession no. ATCC CRL 8006).

Assays for CD20 specificity were accomplished by radioimmunoassay.Briefly, purified anti-CD20 Bl was radiolabeled with I¹²⁵ by theiodobead method as described in Valentine, M. A. et al., (1989) J. Biol.Chem. 264:11282. (I¹²⁵ Sodium Iodide, ICN, Irvine, Calif., Cat. No.28665H). Hybridomas were screened by co-incubation of 0.05 ml of mediafrom each of the fusion wells together with 0.05 ml of I¹²⁵ labeledanti-CD20 Bl (10 ng) in 1% BSA, PBS (pH 7.4), and 0.5 ml of the samebuffer containing 100,000 SB cells. After incubation for 1 hr at roomtemperature, the cells were harvested by transferring to 96 well titerplates (V&P Scientific, San Diego, Calif.), and washed thoroughly.Duplicate wells containing unlabeled anti-CD20 Bl and wells containingno inhibiting antibody were used as positive and negative controls,respectively. Wells containing greater than 50% inhibition were expandedand cloned. The antibody demonstrating the highest inhibition wasderived from the cloned cell line designated herein as "2B8."

B. Preparation of 2B8-MX-DTPA Conjugate

i. MX-DTPA

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid ("carbon-14 labeled MX-DTPA") was used as achelating agent for conjugation of radiolabel to 2B8. Manipulations ofMX-DTPA were conducted to maintain metal-free conditions, ie, metal-freereagents were utilized and, when possible, polypropylene plasticcontainers (flasks, beakers, graduated cylinders, pipette tips) washedwith ALCONOX® (a detergent) and rinsed with MILLI-Q® water (purifiedwater), were similarly utilized. MX-DTPA was obtained as a dry solidfrom Dr. Otto Gansow (National Institute of Health, Bethesda, Md.) andstored desiccated at 4° C. (protected from light), with stock solutionsbeing prepared in Milli-Q® water at a concentration of 2-5 mM, withstorage at -70° C. MX-DTPA was also obtained from Coulter Immunology(Hialeah, Fla.) as the disodium salt in water and stored at -70° C.

ii. Preparation of 2B8

Purified 2B8 was prepared for conjugation with MX-DTPA by transferringthe antibody into metal-free 50 mM bicine-NaOff, pH 8.6, containing 150mM NaCl, using repetitive buffer exchange with CENTRICON 30™ spinfilters (30,000D, MWCO; Amicon). Generally, 50-200 μL of protein (10mg/nl) was added to the filter unit, followed by 2 mL of bicine buffer.The filter was centrifuged at 4° C. in a Sorval SS-34 rotor (6,000 rpm,45 min.). Retentate volume was approximately 50-100 μL; this process wasrepeated twice using the same filter. Retentate was transferred to apolypropylene 1.5 mL screw cap tube, assayed for protein, diluted to10.0 mg/mL and stored at 4° C. until utilized; protein was similarlytransferred into 50 mM sodium citrate, pH 5.5, containing 150 mM NaCland 0.05% sodium azide, using the foregoing protocol.

iii. Conjugation of 2B8 with MX-DTPA

Conjugation of 2B8 with MX-DTPA was performed in polypropylene tubes atambient temperature. Frozen MX-DTPA stock solutions were thawedimmediately prior to use. 50-200 mL of protein at 10 mg/mL were reactedwith MX-DTPA at a molar ratio of MX-DTPA-to-2B8 of 4:1. Reactions wereinitiated by adding the MX-DTPA stock solution and gently mixing; theconjugation was allowed to proceed overnight (14 to 20 hr), at ambienttemperature. Unreacted MX-DTPA was removed from the conjugate bydialysis or repetitive ultrafiltration, as described above in ExampleI.B.ii, into metal-free normal saline (0.9% w/v) containing 0.05% sodiumazide. The protein concentration was adjusted to 10 mg/mL and stored at4° C. in a polypropylene tube until radiolabeled.

iv. Determination of MX-DTPA Incorporation

MX-DTPA incorporation was determined by scintillation counting andcomparing the value obtained with the purified conjugate to the specificactivity of the carbon- 14!-labeled MX-DTPA. For certain studies, inwhich non-radioactive MX-DTPA (Coulter Immunology) was utilized, MX-DTPAincorporation was assessed by incubating the conjugate with an excess ofa radioactive carrier solution of yttrium- 90! of known concentrationand specific activity.

A stock solution of yttrium chloride of known concentration was preparedin metal-free 0.05N HCl to which carrier-free yttrium- 90! (chloridesalt) was added. An aliquot of this solution was analyzed by liquidscintillation counting to determine an accurate specific activity forthis reagent. A volume of the yttrium chloride reagent equal to 3-timesthe number of mols of chelate expected to be attached to the antibody,(typically 2 mol/mol antibody), was added to a polypropylene tube, andthe pH adjusted to 4.0-4.5 with 2M sodium acetate. Conjugated antibodywas subsequently added and the mixture incubated 15-30 min. at ambienttemperature. The reaction was quenched by adding 20 mM EDTA to a finalconcentration of 1 mM and the pH of the solution adjusted toapproximately pH 6 with 2M sodium acetate.

After a 5 min. incubation, the entire volume was purified byhigh-performance, size-exclusion chromatography (described infra). Theeluted protein-containing fractions were combined, the proteinconcentration determined, and an aliquot assayed for radioactivity. Thechelate incorporation was calculated using the specific activity of theyttrium- 90! chloride preparation and the protein concentration.

v. Immunoreactivity of 2B8-MX-DTPA

The immunoreactivity of conjugated 2B8 was assessed using whole-cellELISA. Mid-log phase SB cells were harvested from culture bycentrifugation and washed two times with 1X HBSS. Cells were diluted to1-2×10⁶ cells/mL in HBSS and aliquoted into 96-well polystyrenemicrotiter plates at 50,000-100,000 cells/well. The plates were driedunder vacuum for 2 h. at 40°-45° C. to fix the cells to the plastic;plates were stored dry at -20° C. until utilized. For assay, the plateswere warmed to ambient temperature immediately before use, then blockedwith 1× PBS, pH 7.2-7.4 containing 1% BSA (2 h). Samples for assay werediluted in 1× PBS/1% BSA, applied to plates and serially diluted (1:2)into the same buffer. After incubating plates for 1 h. at ambienttemperature, the plates were washed three times with 1× PBS. Secondaryantibody (goat anti-mouse IgG1-specific HRP conjugate 50 μL) was addedto wells (1:1500 dilution in 1× PBS/1% BSA) and incubated 1 h. atambient temperature. Plates were washed four times with 1× PBS followedby the addition of ABTS substrate solution (50 mM sodium citrate, pH 4.5containing 0.01% ATBS and 0.001% H202). Plates were read at 405 nm after15-30 min. incubation. Antigen-negative HSB cells were included inassays to monitor non-specific binding. Immunoreactivity of theconjugate was calculated by plotting the absorbance values vs. therespective dilution factor and comparing these to values obtained usingnative antibody (representing 100% immunoreactivity) tested on the sameplate; several values on the linear portion of the titration profilewere compared and a mean value determined (data not shown).

vi. Preparation of Indium- 111!-Labeled 2B8-MX-DTPA ("I2B8")

Conjugates were radiolabeled with carrier-free indium- 111!. An aliquotof isotope (0.1-2 mCi/mg antibody) in 0.05M HCL was transferred to apolypropylene tube and approximately one-tenth volume of metal-free 2MHCl added. After incubation for 5 min., metal-free 2M sodium acetate wasadded to adjust the solution to pH 4.0-4.4. Approximately 0.5 mg of2B8-MX-DTPA was added from a stock solution of 10.0 mg/mL DTPA in normalsaline, or 50 mM sodium citrate/150 mM NaCl containing 0.05% sodiumazide, and the solution gently mixed immediately. The pH solution waschecked with pH paper to verify a value of 4.0-4.5 and the mixtureincubated at ambient temperature for 15-30 min. Subsequently, thereaction was quenched by adding 20 mM EDTA to a final concentration of 1mM and the reaction mixture was adjusted to approximately pH 6.0 using2M sodium acetate.

After a 5-10 min. incubation, uncomplexed radioisotope was removed bysize-exclusion chromatography. The HPLC unit consisted of Waters Model6000 or TosoHaas Model TSK-6110 solvent delivery system fitted,respectively, with a Waters U6K or Rheodyne 700 injection valve.Chromatographic separations were performed using a gel permeation column(BioRad SEC-250; 7.5×300 mm or comparable TosoHaas column) and a SEC-250guard column (7.5×100 mm). The system was equipped with a fractioncollector (Pharmacia Frac200) and a UV monitor fitted with a 280 nmfilter (Pharmacia model UV-1). Samples were applied and elutedisocratically using 1× PBS, pH 7.4, at 1.0 mL/min flow rate. One-halfmilliliter fractions were collected in glass tubes and aliquots of thesecounted in a gamma counter. The lower and upper windows were set to 100and 500 KeV respectively.

The radioincorporation was calculated by summing the radioactivityassociated with the eluted protein peak and dividing this number by thetotal radioactivity eluted from the column; this value was thenexpressed as a percentage (data not shown). In some cases, theradioincorporation was determined using instant thin-layerchromatography ("ITLC"). Radiolabeled conjugate was diluted 1:10 or 1:20in 1× PBS containing or 1× PBS/1 mM DTPA, then 1 μL was spotted 1.5 cmfrom one end of a 1×5 cm strip of ITLC SG paper. The paper was developedby ascending chromatography using 10% ammonium acetate in methanol:water(1:1;v/v). The strip was dried, cut in half crosswise, and theradioactivity associated with each section determined by gamma counting.The radioactivity associated with the bottom half of the strip(protein-associated radioactivity) was expressed as a percentage of thetotal radioactivity, determined by summing the values for both top andbottom halves (data not shown).

Specific activities were determined by measuring the radioactivity of anappropriate aliquot of the radiolabeled conjugate. This value wascorrected for the counter efficiency (typically 75%) and related to theprotein concentration of the conjugate, previously determined byabsorbance at 280 nm, and the resulting value expressed as mCi/mgprotein.

For some experiments, 2B8-MX-DTPA was radiolabeled with indium 111!following a protocol similar to the one described above but withoutpurification by HPLC; this was referred to as the "mix-and-shoot"protocol.

vii. Preparation of Yttrium- 90!-Labeled 2B8-M-DTPA ("Y2B8")

The same protocol described for the preparation of I2B8 was followed forthe preparation of the yttrium- 90!-labeled 2B8-MX-DTPA ("Y2B8")conjugate except that 2 ng HCl was not utilized; all preparations ofyttrium-labeled conjugates were purified by size-exclusionchromatography as described above.

C. Non-Human Animal Studies.

i. Biodistribution of Radiolabeled 2B8-MX-DTPA

I2B8 was evaluated for tissue biodistribution in six-to-eight week oldBALB/c mice. The radiolabeled conjugate was prepared usingclinical-grade 2B8-MX-DTPA following the "mix and shoot" protocoldescribed above. The specific activity of the conjugate was 2.3 mCi/mgand the conjugate was formulated in PBS, pH 7.4 containing 50 mg/mL HSA.Mice were injected intravenously with 100 μL of I2B8 (approximately 21μCi) and groups of three mice were sacrificed by cervical dislocation at0, 24, 48, and 72 hours. After sacrifice, the tail, heart, lungs, liver,kidney, spleen, muscle, and femur were removed, washed and weighed; asample of blood was also removed for analysis. Radioactivity associatedwith each specimen was determined by gamma counting and the percentinjected dose par gram tissue subsequently determined. No attempt wasmade to discount the activity contribution represented by the bloodassociated with individual organs.

In a separate protocol, aliquots of 2B8-MX-DTPA incubated at 4° C. and30° C. for 10 weeks were radiolabeled with indium- 111! to a specificactivity of 2.1 mCi/mg for both preparations. These conjugates were thenused in biodistribution studies in mice as described above.

For dosimetry determinations, 2B8-MX-DTPA was radiolabeled with indium-111! to a specific activity of 2.3 mCi/mg and approximately 1.1μ Ci wasinjected into each of 20 BALB/c mice. Subsequently, groups of five miceeach were sacrificed at 1, 24, 48 and 72 hours and their organs removedand prepared for analysis. In addition, portions of the skin, muscle andbone were removed and processed for analysis; the urine and feces werealso collected and analyzed for the 24-72 hour time points.

Using a similar approach, 2B8-MX-DTPA was also radiolabeled withyttrium- 90! and its biological distribution evaluated in BALB/c miceover a 72-hour time period. Following purification by HPLC sizeexclusion chromatography, four groups of five mice each were injectedintravenously with approximately 1μ Ci of clinically-formulatedconjugate (specific activity:12.2 mCi/mg); groups were subsequentlysacrificed at 1, 24, 48 and 72 hours and their organs and tissuesanalyzed as described above. Radioactivity associated with each tissuespecimen was determined by measuring bremstrahlung energy with a gammascintillation counter. Activity values were subsequently expressed aspercent injected dose per gram tissue or percent injected dose perorgan. While organs and other tissues were rinsed repeatedly to removesuperficial blood, the organs were not perfused. Thus, organ activityvalues were not discounted for the activity contribution represented byinternally associated blood.

ii. Tumor Localization of I2B8

The localization of radiolabeled 2B8-MX-DTPA was determined in athymicmice bearing Ramos B cell tumors. Six-to-eight week old athymic micewere injected subcutaneously (left-rear flank) with 0.1 mL of RPMI-1640containing 1.2×10⁷ Ramos tumor cells which had been previously adaptedfor growth in athymic mice. Tumors arose within two weeks and ranged inweight from 0.07 to 1.1 grams. Mice were injected intravenously with 100μL of indium- 111!-labeled 2B8-MX-DTPA (16.7μ Ci) and groups of threemice were sacrificed by cervical dislocation at 0, 24, 48, and 72 hours.After sacrifice the tail, heart, lungs, liver, kidney, spleen, muscle,femur, and tumor were removed, washed, weighed; a sample of blood wasalso removed for analysis. Radioactivity associated with each specimenwas determined by gamma counting and the percent injected dose per gramtissue determined.

iii. Biodistribution and Tumor Localization Studies with Radiolabeled2B8-MX-DTPA

Following the preliminary biodistribution experiment described above(Example I.B.viii.a.), conjugated 2B8 was radiolabeled with indium- 111!to a specific activity of 2.3 mCi/mg and roughly 1.1μ Ci was injectedinto each of twenty BALB/c mice to determine biodistribution of theradiolabeled material. Subsequentially, groups of five mice each weresacrificed at 1, 24, 48 and 72 hours and their organs and a portion ofthe skin, muscle and bone were removed and processed for analysis. Inaddition, the urine and feces were collected and analyzed for the 24-72hour time-points. The level of radioactivity in the blood dropped from40.3% of the injected dose per gram at 1 hour to 18.9% at 72 hours (datanot shown). Values for the heart, kidney, muscle and spleen remained inthe range of 0.7-9.8% throughout the experiment. Levels of radioactivityfound in the lungs decreased from 14.2% at 1 hour to 7.6% at 72 hours;similarly the respective liver injected-dose per gram values were 10.3%and 9.9%. These data were used in determining radiation absorbed doseestimates I2B8 described below.

The biodistribution of yttrium- 90!-labeled conjugate, having a specificactivity of 12.2 mCi/mg antibody, was evaluated in BALB/c mice.Radioincorporations of >90% were obtained and the radiolabeled antibodywas purified by HPLC. Tissue deposition of radioactivity was evaluatedin the major organs, and the skin, muscle, bone, and urine and fecesover 72 hours and expressed as percent injected dose/g tissue. Results(not shown) evidenced that while the levels of radioactivity associatedwith the blood dropped from approximately 39.2% injected dose per gramat 1 hour to roughly 15.4% after 72 hours the levels of radioactivityassociated with tail, heart, kidney, muscle and spleen remained fairlyconstant at 10.2% or less throughout the course of the experiment.Importantly, the radioactivity associated with the bone ranged from 4.4%of the injected dose per gram bone at 1 hour to 3.2% at 72 hours. Takentogether, these results suggest that little free yttrium was associatedwith the conjugate and that little free radiometal was released duringthe course of the study. These data were used in determining radiationabsorbed dose estimates for Y2B8 described below.

For tumor localization studies, 2B8-MX-DTPA was prepared andradiolabeled with ¹¹¹ Indium to a specific activity of 2.7 mCi/mg. Onehundred microliters of labeled conjugate (approximately 24μ Ci) weresubsequently injected into each of 12 athymic mice bearing Ramos B celltumors. Tumors ranged in weight from 0.1 to 1.0 grams. At time points of0, 24, 48, and 72 hours following injection, 50 μL of blood was removedby retro-orbital puncture, the mice sacrificed by cervical dislocation,and the tail, heart, lungs, liver, kidney, spleen, muscle, femur, andtumor removed. After processing and weighing the tissues, theradioactivity assiocated with each tissue specimen was determined usinga gammma counter and the values expressed as percent injected dose pergram.

The results (not shown) evidenced that the tumor concentrations of the¹¹¹ In-2B8-MX-DTPA increased steadily throughout the course of theexperiment. Thirteen percent of the injected dose was accumulated in thetumor after 72 hours. The blood levels, by contrast, dropped during theexperiment from over 30% at time zero to 13% at 72 hours. All othertissues (except muscle) contained between 1.3 and 6.0% of the injecteddose per gram tissue by the end of the experiment; muscle tissuecontained approximately 13% of the injected dose per gram.

D. Humnan Studies

i. 2B8 and 2B8-MX-DTPA: Immunohistology Studies with Human Tissues

The tissue reactivity of murine monoclonal antibody 2B8 was evaluatedusing a panel of 32 different human tissues fixed with acetone. Antibody2B8 reacts with the anti-CD20 antigen which had a very restrictedpattern of tissue distribution, being observed only in a subset of cellsin lymphoid tissues including those of hematopoietic origin.

In the lymph node, immunoreactivity was observed in a population ofmature cortical B-lymphocytes as well as proliferating cells in thegerminal centers. Positive reactivity was also observed in theperipheral blood, B-cell areas of the tonsils, white pulp of the spleen,and with 40-70% of the medullary lymphocytes found in the thymus.Positive reactivity was also seen in the follicles of the lamina propria(Peyer's Patches) of the large intestines. Finally, aggregates orscattered lymphoid cells in the stroma of various organs, including thebladder, breast, cervix, esophagus, lung, parotid, prostate, smallintestine, and stomach, were also positive with antibody 2B8 (data notshown).

All simple epithelial cells, as well as the stratified epithelia andepithelia of different organs, were found to be unreactive. Similarly,no reactivity was seen with neuroectodermal cells, including those inthe brain, spinal cord and peripheral nerves. Mesenchymal elements, suchas skeletal and smooth muscle cells, fibroblasts, endothelial cells, andpolymorphonuclear inflammatory cells were also found to be negative(data not shown).

The tissue reactivity of the 2B8-MX-DTPA conjugate was evaluated using apanel of sixteen human tissues which had been fixed with acetone. Aspreviously demonstrated with the native antibody (data not shown), the2B8-MX-DTPA conjugate recognized the CD20 antigen which exhibited ahighly restricted pattern of distribution, being found only on a subsetof cells of lymphoid origin. In the lymph node, immunoreactivity wasobserved in the B cell population. Strong reactivity was seen in thewhite pulp of the spleen and in the medullary lymphocytes of the thymus.Immunoreactivity was also observed in scattered lymphocytes in thebladder, heart, large intestines, liver, lung, and uterus, and wasattributed to the presence of inflammatory cells present in thesetissues. As with the native antibody, no reactivity was observed withneuroectodermal cells or with mesenchymal elements (data not shown).

ii. Clinical Analysis of I2B8 (Imaging) and Y2B8 (Therapy)

a. Phase I/II Clinical Trial Single Dose Therapy Study

A Phase I/II clinical analysis of I2B8 (imaging) followed by treatmentwith a single therapeutic dose of Y2B8 is currently being conducted,

For the single-dose study, the following schema is being followed:

1. Peripheral Stem Cell (PSC) or Bone Marrow (BM) Harvest with Purging;

2. I2B8 Imaging;

3. Y2B8 Therapy (three Dose Levels); and

4. PSC or Autologous BM Transplantation (if necessary based uponabsolute neutrophil count below 500/mm³ for three consecutive days orplatelets below 20,000/mm³ with no evidence of marrow recovery on bonemarrow examination).

The Dose Levels of Y2B8 are as follows:

    ______________________________________                                        Dose Level    Dose (mCi)                                                      ______________________________________                                        1.            20                                                              2.            30                                                              3.            40                                                              ______________________________________                                    

Three patients are to be treated at each of the dose levels fordetermination of a Maximum Tolerated Dose ("MTD").

Imaging (Dosimetry) Studies are conducted as follows: each patient isinvolved in two in vivo biodistribution studies using I2B8. In the firststudy, 2 mg of I2B8 (5 mCi), is administered as an intravenous (i.v.)infusion over one hour; one week later 2B8 (ie, unconjugated antibody)is administered by i.v. at a rate not to exceed 250 mg/hr followedimmediately by 2 mg of I2B8 (5 mCi) administered by i.v. over one hour.In both studies, immediately following the I2B8 infusion, each patientis imaged and imaging is repeated at time t=14-18 hr (if indicated),t=24 hr; t=72 hr; and t=96 hr (if indicated). Whole body averageretention times for the indium 111! label are determined; suchdeterminations are also made for recognizable organs or tumor lesions("regions of interest").

The regions of interest are compared to the whole body concentrations ofthe label; based upon this comparison, an estimate of the localizationand concentration of Y2B8 can be determined using standard protocols. Ifthe estimated cumulative dose of Y2B8 is greater than eight (8) timesthe estimated whole body dose, or if the estimated cumulative dose forthe liver exceeds 1500 _(c) Gy, no treatment with Y2B8 should occur.

If the imaging studies are acceptible, either 0.0 or 1.0 mg/kg patientbody weight of 2B8 is administered by i.v. infusion at a rate not toexceed 250 mg/h. This is followed by administration of Y2B8 (10,20 or 40mCi) at an i.v. infusion rate of 20 mCi/hr.

b. Phase I/II Clinical Trial: Multiple Dose Therapy Study

A Phase I/II clinical analysis of Y2B8 is currently being conducted. Forthe multiple-dose study, the following schema is being followed:

1. PSC or BM Harvest;

2. I2B8 Imaging;

3. Y2B8 Therapy (three Dose Levels) for four doses or a total cumulativedose of 80 mCi; and

4. PSC or Autologous BM Transplantation (based upon decision of medicalpractitioner).

The Dose Levels of Y2B8 are as follows:

    ______________________________________                                        Dose Level    Dose (mCi)                                                      ______________________________________                                        1.            10                                                              2.            15                                                              3.            20                                                              ______________________________________                                    

Three patients are to be treated at each of the dose levels fordetermination of an MTD.

Imaging (Dosimetry) Studies are conducted as follows: A preferredimaging dose for the unlabeled antibody (ie 2B8) will be determined withthe first two patients. The first two patients will receive 100 mg ofunlabeled 2B8 in 250 cc of normal saline over 4 hrs followed by 0.5 mCiof I2B8--blood will be sampled for biodistribution data at times t=0,t=10 min., t=120 min., t=24 hr, and t=48 hr. Patients will be scannedwith multiple regional gamma camera images at times t=2 hr, t=24 hr andt=48 hr. After scanning at t=48 hr, the patients will receive 250 mg of2B8 as described, followed by 4.5 mCi of I2B8--blood and scanning willthen follow as described. If 100 mg of 2B8 produces superior imaging,then the next two patients will receive 50 mg of 2B8 as described,followed by 0.5 mCi of I2B8 followed 48 hrs later by 100 mg 2B8 and thenwith 4.5 mCi of I2B8. If 250 mg of 2B8 produces superior imaging, thenthe next two patients will receive 250 mg of 2B8 as described, followedby 0.5 mCi of I2B8 followed 48 hrs later with 500 mg 2B8 and then with4.5 mCi of I2B8. Subsequent patients will be treated with the lowestamount of 2B8 that provides optimal imaging. Optimal imaging will bedefined by: (1) best effective imaging with the slowest disappearance ofantibody; (2) best distribution minimizing compartmentalization in asingle organ; and (3) best subjective resolution of the lesion(tumor/background comparison).

For the first four patients, the first therapeutic dose of Y2B8 willbegin 14 days after the last dose of I2B8; for subsequent patients, thefirst therapeutic dose of Y2B8 will begin between two to seven daysafter the I2B8.

Prior to treatment with Y2B8, for the patients other than the firstfour, 2B8 will be administered as described, followed by i.v. infusionof Y2B8 over 5-10 min. Blood will be sampled for biodistribution attimes t=0, t=10 min., t=120 min., t=24 hr and t=48 hr. Patients willreceive repetitive doses of Y2B8 (the same dose administered as with thefirst dose) approximately every six to eight weeks for a maximum of fourdoses, or total cumulative dose of 80 mCi. It is most preferred thatpatients not receive a subsequent dose of Y2B8 until the patients' WBCis greater than/equal to 3,000 and AGC is greater than/equal to 100,000.

Following completion of the three-dose level study, an MTD will bedefined. Additional patients will then be enrolled in the study andthese will receive the MTD.

II. CHIMERIC ANTI-CD20 ANTIBODY PRODUCTION ("C2B8")

A. Construction of Chimeric Anti-CD20 Immunoglobulin DNA ExpressionVector

RNA was isolated from the 2B8 mouse hybridoma cell (as described inChomczynki, P. et al., "Single step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction." Anal. Biochem.162:156-159 (1987)). and cDNA was prepared therefrom. The mouseimmunoglobulin light chain variable region DNA was isolated from thecDNA by polymerase chain reaction using a set of DNA primers withhomology to mouse light chain signal sequences at the 5' end and mouselight chain J region at the 3' end. Primer sequences were as follows:

1. V_(L) Sense (SEQ. ID. NO. 3)

5' ATC AC AGATCT CTC ACC ATG GAT TTT CAG GTG CAG ATT ATC AGC TTC 3'

(The underlined portion is a Bgl II site; the above-lined portion is thestart codon.)

2. V_(L) Antisense (SEQ. ID. NO. 4)

5' TGC AGC ATC CGTACG TTT GAT TTC CAG CTT 3'

(The underlined portion is a Bsi WI site.)

See, FIGS. 1 and 2A-E for the corresponding Bgl II and Bsi WI sites inTCAE 8, and FIGS. 3A-F for the corresponding sites in anti-CD20 in TCAE8.

These resulting DNA fragment was cloned directly into the TCAE 8 vectorin front of the human kappa light chain constant domain and sequenced.The determined DNA sequence for the murine variable region light chainis set forth in FIGS. 3A-F (SEQ. ID. NO. 5); see also FIG. 3,nucleotides 978 through 1362. FIG. 4 further provides the amino acidsequence from this murine variable region, and the CDR and frameworkregions. The mouse light chain variable region from 2B8 is in the mousekappa VI family. See, Kabat, supra.

The mouse heavy chain variable region was similarly isolated and clonedin front of the human IgGl constant domains. Primers were as follows:p 1. V_(H) Sense (SEQ. ID. NO. 6)

5' GCG GCT CCC ACGCGT GTC CTG TCC CAG 3'

(The underlined portion is an Mlu I site.)

2. V_(H) Antisense (SEQ. ID. NO. 7)

5' GG(G/C) TGT TGT GCTAGC TG(A/C) (A/G)GA GAC (G/A)GT GA 3'

(The underlined portion is an Nhe I site.)

See, FIGS. 1 and 2A-E for corresponding Mlu I and Nhe I sites in TCAE 8,and FIGS. 3A-F for corresponding sites in anti-CD20 in TCAE 8.

The sequence for this mouse heavy chain is set forth in FIG. 5 (SEQ. ID.NO. 8); see also FIGS. 3A-F, nucleotide 2401 through 2820. FIG. 5 alsoprovides the amino acid sequence from this murine variable region, andthe CDR and framework regions. The mouse heavy chain variable regionfrom 2B8 is in the mouse VH 2B family. See, Kabat, supra.

B. Creation of Chimeric Anti- CD20 Producing CHO and SP2/0 Transfectomas

Chinese hamster ovary ("CHO") cells DG44 were grown in SSFM II minushypoxanthine and thymidine media (Gibco, Grand Island, N.Y., Form No.91-0456PK); SP2/0 mouse myeloma cells were grown in Dulbecco's ModifiedEagles Medium media ("DMEM") (Irvine Scientific, Santa Ana, Calif., Cat.No. 9024) with 5% fetal bovine serum and 20 ml/L glutamine added. Fourmillion cells were electroporated with either 25 μg CHO or 50 μg SP2/0plasmid DNA that had been restricted with Not I using a BTX 600electroporation system (BTX, San Diego, Calif.) in 0.4 ml disposablecuvettes. Conditions were either 210 volts for CHO or 180 volts forSP2/0, 400 microfaradays, 13 ohms. Each electroporation was plated intosix 96 well dishes (about 7,000 cells/well). Dishes were fed with mediacontaining G418 (GENETICIN, Gibco, Cat. No. 860-1811) at 400 μg/mlactive compound for CHO (media further included 50 μM hypoxanthine and 8μM thymidine) or 800 μg/ml for SP2/0, two days following electroporationand thereafter 2 or 3 days until colonies arose. Supernatant fromcolonies was assayed for the presence of chimeric immunoglobulin via anELISA specific for human antibody. Colonies producing the highest amountof immunoglobulin were expanded and plated into 96 well platescontaining media plus methotrexate (25 nM for SP2/0 and 5 nM for CHO)and fed every two or three days. Supernatants were assayed as above andcolonies producing the highest amount of immunoglobulin were examined.Chimeric anti-CD20 antibody was purified from supernatant using proteinA affinity chromatography.

Purified chimeric anti-CD20 was analyzed by electrophoresis inpolyacrylamide gels and estimated to be greater than about 95% pure.Affinity and specificity of the chimeric antibody was determined basedupon 2B8. Chimeric anti-CD20 antibody tested in direct and competitivebinding assays, when compared to murine anti-CD20 monoclonal antibody2B8, evidenced comparable affinity and specificity on a number of CD20positive B cells lines (data not presented). The apparent affinityconstant ("Kap") of the chimeric antibody was determined by directbinding of I¹²⁵ radiolabeled chimeric anti-CD20 and compared toradiolabeled 2B8 by Scatchard plot; estimated Kap for CHO producedchimeric anti-CD20 was 5.2×10⁻⁹ M and for SP2/0 produced antibody,7.4×10⁻⁹ M. The estimated Kap for 2B8 was 3.5×10⁻⁹ M. Direct competitionby radioimmunoassay was utilized to confirm both the specificity andretention of immunoreactivity of the chimeric antibody by comparing itsability to effectively compete with 2B8. Substantially equivalentamounts of chimeric anti-CD20 and 2B8 antibodies were required toproduce 50% inhibition of binding to CD20 antigens on B cells (data notpresented), ie, there was a minimal loss of inhibiting activity of theanti-CD20 antibodies, presumably due to chimerization.

The results of Example II.B indicate, inter alia, that chimericanti-CD20 antibodies were generated from CHO and SP2/0 transfectomasusing the TCAE 8 vectors, and these chimeric antibodies hadsubstantially the same specificity and binding capability as murineanti-CD20 monoclonal antibody 2B8.

C. Determination of Immunological Activity of Chimeric Anti-CD20Antibodies

i. Human C1q Analysis

Chimeric anti-CD20 antibodies produced by both CHO and SP2/0 cell lineswere evaluated for human C1q binding in a flow cytometry assay usingfluorescein labeled C1q (C1q was obtained from Quidel, Mira Mesa,Calif., Prod. No. A400 and FITC label from Sigma, St. Louis Mo., Prod.No. F-7250; FITC. Labeling of C1q was accomplished in accordance withthe protocol described in Selected Methods In Cellular Immunology,Michell & Shiigi, Ed. (W. H. Freeman & Co., San Francisco, Calif., 1980,p. 292). Analytical results were derived using a Becton DickinsonFACScan™ flow cytometer (fluorescein measured over a range of 515-545nm). Equivalent amounts of chimeric anti-CD20 antibody, human IgG1,Kmyeloma protein (Binding Site, San Diego, Calif., Prod. No. BP078), and2B8 were incubated with an equivalent number of CD20-positive SB cells,followed by a wash step with FACS buffer (0.2% BSA in PBS, pH 7.4, 0.02%sodium azide) to remove unattached antibody, followed by incubation withFITC labeled C1q. Following a 30-60 min. incubation, cells were againwashed. The three conditions, including FITC-labeled C1q as a control,were analyzed on the FACScan™ following manufacturing instructions.Results are presented in FIG. 6.

As the results of FIG. 6 evidence, a significant increase influorescence was observed only for the chimeric anti-CD20 antibodycondition; ie, only SB cells with adherent chimeric anti-CD20 antibodywere C1q positive, while the other conditions produced the same patternas the control.

ii. Complement Dependent Cell Lvses

Chimeric anti-CD20 antibodies were analyzed for their ability to lyselymphoma cell lines in the presence of human serum (complement source).CD20 positive SB cells were labeled with ⁵¹ Cr by admixing 100μ Ci of ⁵¹Cr with 1×10⁶ SB cells for 1 hr at 37° C.; labeled SB cells were thenincubated in the presence of equivalent amounts of human complement andequivalent amounts (0-50 μg/ml) of either chimeric anti-CD20 antibodiesor 2B8 for 4 hrsat 37° C. (see, Brunner, K. T. et al., "Quantitativeassay of the lytic action of immune lymphoid cells on ⁵¹ Cr-labeledallogeneic target cells in vitro." Immunology 14:181-189 (1968). Resultsare presented in FIG. 7.

The results of FIG. 7 indicate, inter alia, that chimeric anti-CD20antibodies produced significant lysis (49%) under these conditions.

ii. Antibody Dependent Cellular Cytotoxicity Effector Assay

For this study, CD20 positive cells (SB) and CD20 negative cells (T cellleukemia line HSB; see, Adams, Richard, "Formal Discussion," Can. Res.27:2479-2482 (1967); ATCC deposit no. ATCC CCL 120.1) were utilized;both were labeled with ⁵¹ Cr. Analysis was conducted following theprotocol described in Brunner, K. T. et al., "Quantitative assay of thelytic action of immune lymphoid cells on ⁵¹ Cr-labeled allogeneic targetcells in vitro; inhibition by isoantibody and drugs." Immunology14:181-189 (1968); a substantial chimeric anti-CD20 antibody dependentcell mediated lysis of CD20 positive SB target cells (⁵¹ Cr-labeled) atthe end of a 4 hr, 37° C. incubation, was observed and this effect wasobserved for both CHO and SP2/0 produced antibody (effector cells werehuman peripheral lymphocytes; ratio of effector cells:target was 100:1).Efficient lysis of target cells was obtained at 3.9 μg/ml. In contrast,under the same conditions, the murine anti-CD20 monoclonal antibody 2B8had a statistically insignificant effect, and CD20 negative HSB cellswere not lysed. Results are presented in FIG. 8.

The results of Example II indicate, inter alia, that the chimericanti-CD20 antibodies of Example I were immunologically active.

III. DEPLETION OF B CELLS IN VIVO USING CHIMERIC ANTI-CD20

A Non-Human Primate Study

Three separate non-human primate studies were conducted. Forconvenience, these are referred to herein as "Chimeric Anti-CD20: CHO &SP2/0;" "Chimeric Anti-CD20: CHO;" and "High Dosage Chimeric Anti-CD20."Conditions were as follows:

Chimeric Anti-CD20: CHO & SP2/0

Six cynomolgus monkeys ranging in weight from 4.5 to 7 kilograms (WhiteSands Research Center, Alamogordo, N.Mex.) were divided into threegroups of two monkeys each. Both animals of each group received the samedose of immunologically active chimeric anti-CD20 antibody. One animalin each group received purified antibody produced by the CHOtransfectoma; the other received antibody produced by the SP2/0transfectoma. The three groups received antibody dosages correspondingto 0.1 mg/kg, 0.4 mg/kg, and 1.6 mg/kg each day for four (4) consecutivedays. The chimeric immunologically active anti-CD20 antibody, which wasadmixed with sterile saline, was administered by intravenous infusion;blood samples were drawn prior to each infusion. Additional bloodsamples were drawn beginning 24 hrs after the last injection (T=O) andthereafter on days 1, 3, 7, 14 and 28; blood samples were also takenthereafter at biweekly intervals until completion of the study at day90.

Approximately 5 ml of whole blood from each animal was centrifuged at2000 RPM for 5 min. Plasma was removed for assay of soluble chimericanti-CD20 antibody levels. The pellet (containing peripheral bloodleukocytes and red blood cells) was resuspended in fetal calf serum forfluorescent-labeled antibody analysis (see, "Fluorescent AntibodyLabeling of Lymphoid Cell Population," infra.).

Chimeric Anti-CD20: CHO

Six cynomolgus monkeys ranging in weight from 4 to 6 kilograms (WhiteSands) were divided into three groups of two monkeys each. All animalswere injected with immunologically active chimeric anti-CD20 antibodiesproduced from the CHO transfectoma (in sterile saline). The three groupswere separated as follows: subgroup 1 received daily intravenousinjections of 0.01 mg/kg of the antibody over a four (4) day period;subgroup 2 received daily intravenous injections of 0.4 mg/kg of theantibody over a four (4) day period; subgroup 3 received a singleintravenous injection of 6.4 mg/kg of the antibody. For all threesubgroups, a blood sample was obtained prior to initiation of treatment;additionally, blood samples were also drawn at T=0, 1, 3, 7, 14 and 28days following the last injection, as described above, and these sampleswere processed for fluorescent labeled antibody analysis (see,"Fluorescent Antibody Labeling," infra.). In addition to peripheralblood B cell quantitation, lymph node biopsies were taken at days 7, 14and 28 following the last injection, and a single cell preparationstained for quantitation of lymphocyte populations by flow cytometry.

High Dosage Chimeric Anti-CD20

Two cynomolgus monkeys (White Sands) were infused with 16.8 mg/kg of theimmunologically active chimeric anti-CD20 antibodies from the CHOtransfectomas (in sterile saline) weekly over a period of fourconsecutive weeks. At the conclusion of the treatment, both animals wereanesthetized for removal of bone marrow; lymph node biopsies were alsotaken. Both sets of tissue were stained for the presence of Blymphocytes using Leu 16 by flow cytometry following the protocoldescribed in Ling, N. R. et al., "B-cell and plasma cell antigens."Leucocyte Typing III White Cell Differentiations Antigens, A. J.McMichael, Ed. (Oxford University Press, Oxford UK, 1987), p. 302.

Fluorescent Antibody Labeling of Lymphoid Cell Population

After removal of plasma, leukocytes were washed twice with HanksBalanced Salt Solution ("HBSS") and resuspended in a plasma equivalentvolume of fetal bovine serum (heat inactivated at 56° C. for 30 min.). A0.1 ml volume of the cell preparation was distributed to each of six(6), 15 ml conical centrifuge tubes Fluorescein labeled monoclonalantibodies with specificity for the human lymphocyte surface markers CD2(AMAC, Westbrook, Me.), CD20 (Becton Dickinson) and human IgM (BindingSite, San Diego, Calif.) were added to 3 of the tubes for identifying Tand B lymphocyte populations. All reagents had previously testedpositive to the corresponding monkey lymphocyte antigens. Chimericanti-CD20 antibody bound to monkey B cell surface CD20 was measured inthe fourth tube using polyclonal goat anti-human IgG coupled withphycoerythrin (AMAC). This reagent was pre-adsorbed on a monkeyIg-sepharose column to prevent cross-reactivity to monkey Ig, thusallowing specific detection and quantitation of chimeric anti-CD20antibody bound to cells. A fifth tube included both anti-IgM andanti-human IgG reagents for double stained B cell population. A sixthsample was included with no reagents for determination ofautofluorescence. Cells were incubated with fluorescent antibodies for30 min., washed and fixed with 0.5 ml of fixation buffer (0.15M NaCl, 1%paraformaldehyde, pH7.4) and analyzed on a Becton Dickinson FACScan™instrument. Lymphocyte populations were initially identified by forwardversus right angle light scatter in a dot-plot bitmap with unlabeledleucocytes. The total lymphocyte population was then isolated by gatingout all other events. Subsequent fluorescence measurements reflectedonly gated lymphocyte specific events.

Depletion of Peripheral Blood B Lymphocytes

No observable difference could be ascertained between the efficacy ofCHO and SP2/0 produced antibodies in depleting B cells in vivo, althougha slight increase in B cell recovery beginning after day 7 for monkeysinjected with chimeric anti-CD20 antibodies derived from CHOtransfectomas at dosage levels 1.6 mg/kg and 6.4 mg/kg was observed andfor the monkey injected with SP2/0 producing antibody at the 0.4 mg/kgdose level. FIGS. 9A, B and C. provide the results derived from thechimeric anti-CD20:CHO & SP2/0 study, with FIG. 9A directed to the 0.4mg/kg dose level; FIG. 9B directed to the 1.6 mg/kg dose level; and FIG.9C directed to the 6.4 mg/kg dose level.

As is evident from FIGS. 9A-C, there was a dramatic decrease (>95%) inperipheral B cell levels after the therapeutic treatment across alltested dose ranges, and these levels were maintained up to seven (7)days post infusion; after this period, B cell recovery began, and, thetime of recovery initiation was independent of dosage levels.

In the Chimeric Anti-CD20: CHO study, a 10-fold lower antibody dosageconcentration (0.01 mg/kg) over a period of four daily injections (0.04mg/kg total) was utilized. FIG. 10 provides the results of this study.This dosage depleted the peripheral blood B cell population toapproximately 50% of normal levels estimated with either theanti-surface IgM or the Leu 16 antibody. The results also indicate thatsaturation of the CD20 antigen on the B lymphocyte population was notachieved with immunologically active chimeric anti-CD20 antibody at thisdose concentration over this period of time for non-human primates; Blymphocytes coated with the antibody were detected in the blood samplesduring the initial three days following therapeutic treatment. However,by day 7, antibody coated cells were undetectable.

Table I summarizes the results of single and multiple doses ofimmunologically active chimeric anti-CD20 antibody on the peripheralblood populations; single dose condition was 6.4 mg/kg; multiple dosecondition was 0.4 mg/kg over four (4) consecutive days (there resultswere derived from the monkeys described above).

                  TABLE I                                                         ______________________________________                                        PERIPHERAL BLOOD POPULATION FROM C2B8 PRIMATE STUDY                           ______________________________________                                        Monkey   Dose     Day        CD2  Anti-Hu IgG                                 ______________________________________                                        A        0.4 mg/kg                                                                              Prebleed   81.5 --                                                   (4 doses)                                                                               0         86.5 0.2                                                            7         85.5 0.0                                                           21         93.3 --                                                            28         85.5 --                                          B        0.4 mg/kg                                                                              Prebleed   81.7 --                                                   (4 doses)                                                                               0         94.6 0.1                                                            7         92.2 0.1                                                           21         84.9 --                                                            28         84.1 --                                          C        6.4 mg/kg                                                                              Prebleed   77.7 0.0                                                  (1 dose)  7         85.7 0.1                                                           21         86.7 --                                                            28         76.7 --                                          D        6.4 mg/kg                                                                              Prebleed   85.7 0.1                                                  (1 dose)  7         94.7 0.1                                                           21         85.2 --                                                            28         85.9 --                                          ______________________________________                                                Anti-Hu IgG +                                                         Monkey  Anti-Hu IgM* Leu-16  % B Cell Depletion                               ______________________________________                                        A       --           9.4     0                                                        0.3          0.0     97                                                       0.1          1.2     99                                                       --           2.1     78                                                       --           4.1     66                                               B       --           14.8    0                                                        0.2          0.1     99                                                       0.1          0.1     99                                                       --           6.9     53                                                       --           8.7     41                                               C       0.2          17.0    0                                                        0.1          0.0     99                                                       --           14.7    15                                                       --           8.1     62                                               D       0.1          14.4    0                                                        0.2          0.0     99                                                       --           9.2     46                                                       --           6.7     53                                               ______________________________________                                         *Double staining population which indicates extent of chimeric antiCD20       coated B cells.                                                          

The data summarized in Table I indicates that depletion of B cells inperipheral blood under conditions of antibody excess occurred rapidlyand effectively, regardless of single or multiple dosage levels.Additionally, depletion was observed for at least seven (7) daysfollowing the last injection, with partial B cell recovery observed byday 21.

Table II summarizes the effect of immunologically active, chimericanti-CD20 antibodies on cell populations of lymph nodes using thetreatment regimen of Table I (4 daily doses of 0.4 mg/kg; 1 dose of 6.4mg/kg); comparative values for normal lymph nodes (control monkey,axillary and inguinal) and normal bone marrow (two monkeys) are alsoprovided.

                  TABLE II                                                        ______________________________________                                        CELL POPULATIONS OF LYMPH NODES                                               ______________________________________                                        Monkey   Dose     Day        CD2  Anti-Hu IgG                                 ______________________________________                                        A        0.4 mg/kg                                                                              7          66.9 --                                                   (4 doses)                                                                              14         76.9 19.6                                                          28         61.6 19.7                                        B        0.4 mg/kg                                                                              7          59.4 --                                                   (4 doses)                                                                              14         83.2  9.9                                                          28         84.1 15.7                                        C        6.4 mg/kg                                                                              7          75.5 --                                                   (1 dose) 14         74.1 17.9                                                          28         66.9 23.1                                        D        6.4 mg/kg                                                                              7          83.8 --                                                   (1 dose) 14         74.1 17.9                                                          28         84.1 12.8                                        ______________________________________                                                Anti-Hu IgG +        % B                                              Monkey  Anti-Hu IgM  Leu-16  Lymphocyte Depletion                             ______________________________________                                        A       7.4          40.1    1                                                        0.8          22.6    44                                                       --           26.0    36                                               B       29.9         52.2    0                                                        0.7          14.5    64                                                       --           14.6    64                                               C       22.3         35.2    13                                                       1.1          23.9    41                                                       --           21.4    47                                               D       12.5         19.7    51                                                       0.2          8.7     78                                                       --           12.9    68                                               ______________________________________                                                                               % B                                                    Anti-Hu IgG +                                                                            Anti-Hu     Lymphocyte                                      CD2    Anti-Hu IgM                                                                              IgM   Leu-16                                                                              Depletion                              ______________________________________                                        Normal Lymph                                                                  Nodes                                                                         Control 1                                                                     Axillary 55.4   25.0       --    41.4  NA                                     Inguinal 52.1   31.2       --    39.5  NA                                     Normal Bone                                                                   Marrow                                                                        Control 2                                                                              65.3   19.0       --    11.4  NA                                     Control 3                                                                              29.8   28.0       --    16.6  NA                                     ______________________________________                                    

The results of Table II evidence effective depletion of B lymphocytesfor both treatment regimens. Table II further indicates that for thenon-human primates, complete saturation of the B cells in the lymphatictissue with immunologically active, chimeric anti-CD20 antibody was notachieved; additionally, antibody coated cells were observed seven (7)days after treatment, followed by a marked depletion of lymph node Bcells, observed on day 14.

Based upon this data, the single High Dosage Chimeric Anti-CD20 studyreferenced above was conducted, principally with an eye towardpharmacology/toxicology determination. Ie this study was conducted toevaluate any toxicity associated with the administration of the chimericantibody, as well as the efficacy of B cell depletion from peripheralblood lymph nodes and bone marrow. Additionally, because the data ofTable II indicates that for that study, the majority of lymph node Bcells were depleted between 7 and 14 days following treatment, a weeklydosing regimen might evidence more efficacious results. Table IIIsummarizes the results of the High Dosage Chimeric Anti-CD20 study.

                  TABLE III                                                       ______________________________________                                        CELL POPULATIONS OF LYMPH NODES AND BONE MARROW                               Lymphocyte Populations (%)                                                    Monkey CD2     CD20.sup.a                                                                            mIgM + anti-C2B8.sup.b                                                                   C2B8.sup.c                                                                           Day.sup.d                            ______________________________________                                        Inguinal Lymph Node                                                           E      90.0    5.3     4.8        6.5    22                                   F      91.0    6.3     5.6        6.3    22                                   G      89.9    5.0     3.7        5.8    36                                   H      85.4    12.3    1.7        1.8    36                                   Bone Marrow                                                                   E      46.7    4.3     2.6        2.8    22                                   F      41.8    3.0     2.1        2.2    22                                   G      35.3    0.8     1.4        1.4    36                                   H      25.6    4.4     4.3        4.4    36                                   ______________________________________                                         .sup.a Indicates population stained with Leu 16.                              .sup.b Indicates double staining population, positive for surface IgM         cells and chimeric antibody coated cells.                                     .sup.c Indicates total population staining for chimeric antibody includin     double staining surface IgM positive cells and single staining (surface       IgM negative) cells.                                                          .sup.d Days after injection of final 16.8 mg/kg dose.                    

Both animals evaluated at 22 days post treatment cessation containedless than 5% B cells, as compared to 40% in control lymph nodes (see,Table II, supra). Similarly, in the bone marrow of animals treated withchimeric anti-CD20 antibody, the levels of CD20 positive cells were lessthan 3% as compared to 11-15% in the normal animals (see, Table II,supra). In the animals evaluated at 36 days post treatment cessation,one of the animals (H) had approximately 12% B cells in the lymph nodeand 4.4% B cells in bone marrow, while the other (G) had approximately5% B cells in the lymph node and 0.8% in the bone marrow--the data isindicative of significant B cell depletion.

The results of Example III, A indicate, inter alia, that low doses ofimmunologically active, chimeric anti-CD20 leads to long-term peripheralblood B cell depletion in primates. The data also indicates thatsignificant depletion of B cell populations was achieved in peripherallymph nodes and bone marrow when repetitive high doses of the antibodywere administered. Continued follow-up on the test animals has indicatedthat even with such severe depletion of peripheral B lymphocytes duringthe first week of treatment, no adverse health effects have beenobserved. Furthermore, as recovery of B cell population was observed, aconclusion to be drawn is that the pluripotent stem cells of theseprimates were not adversely affected by the treatment.

B. Clinical Analysis of C2B8

i. Phase I/II Clinical Trial of C2B8: Single Dose Therapy Study

Fifteen patients having histologically documented relapsed B celllymphoma have been treated with C2B8 in a Phase I/II Clinical Trial.Each patient received a single dose of C2B8 in a dose-escalating study;there were three patients per dose: 10 mg/m² ; 50 mg/m² ; 100 mg/m² ;250 mg/m² and 500 mg/m². Treatment was by i.v. infusion through an 0.22micron in-line filter with C2B8 being diluted in a final volume of 250cc or a maximal concentration of 1 mg/ml of normal saline. Initial ratewas 50 cc/hr for the first hour; if no toxicity was seen, dose rate wasable to be escalated to a maximum of 200 cc/hr.

Toxicity (as indicated by the clinician) ranged from "none", to "fever"to "moderate" (two patients) to "severe" (one patient); all patientscompleted the therapy treatment. Peripheral Blood Lymphocytes wereanalyzed to determine, inter alia, the impact of C2B8 on T-cells andB-cells. Consistently for all patients, Peripheral Blood B Lymphocyteswere depleted after infusion with C2B8 and such depletion was maintainedfor in excess of two weeks.

One patient (receiving 100 mg/² of C2B8) evidenced a Partial Response tothe C2B8 treatment (reduction of greater than 50% in the sum of theproducts of the perpendicular diameters of all measurable indicatorlesions lasting greater than four weeks, during which no new lesions mayappear and no existing lesions may enlarge); at least one other patient(receiving 500 mg/m²) evidenced a Minor Response to the C2B8 treatment(reduction of less than 50% but at least 25% in the sum of the productsof the two longest perpendicular diameters of all measurable indicatorlesions). For presentational efficiency, results of the PBLs are setforth in FIGS. 14A and B; data for the patient evidencing a PR is setforth in FIG. 14; for the patient evidencing an MR, data is set forth inFIG. 14B. In FIGS. 14A and B, the following are applicable:=Lymphocytes; =CD3+cells (T cells); =CD20⁺ cells; =CD19⁺ cells; =Kappa;=lambda; and =C2B8. As evidenced, the B cell markers CD20 and CD 19,Kappa and Lambda, were depleted for a period in excess of two weeks;while there was a slight, initial reduction in T-cell counts, thesereturned to an approximate base-line level in a relatively rapidtime-frame.

ii. Phase I/II Clinical Trial of C2B8: Multiple Dose Therapy Study

Patients having histologically confirmed B cell lymphoma with measurableprogressive disease are eligible for this study which is separated intotwo parts: in Phase I, consisting of a dose escalation to characterizedose limiting toxicities and determination of biologically activetolerated dose level, groups of three patients will receive weekly i.v.infusions of C2B8 for a total of four (4) separate infusions. Cumulativedose at each of the three levels will be as follows: 500 mg/m² (125mg/m² /infusion); 1000 mg/m² (250 mg/m² /infusion);

1500 mg/m² (375 mg/m² /infusion. A biologically active tolerated dose isdefined, and will be determined, as the lowest dose with both tolerabletoxicity and adequate activity); in Phase II, additional patients willreceive the biologically active tolerated dose with an emphasis ondetermining the activity of the four doses of C2B8.

IV. COMBINATION THERAPY: C2B8 AND Y2B8

A combination therapeutic approach using C2B8 and Y2B8 was investigatedin a mouse xenographic model (nu/nu mice, female, approximately 10 weeksold) utilizing a B cell lymphoblastic tumor (Ramos tumor cells). Forcomparative purposes, additional mice were also treated with C2B8 andY2B8.

Ramos tumor cells (ATCC, CRL 1596) were maintained in culture usingRPMI-1640 supplemented with 10% fetal calf serum and glutamine at 37° C.and 5% CO₂. Tumors were initiated in nine female nude mice approximately7-10 weeks old by subcutaneous injection of 1.7×10⁶ Ramos cells in avolume of 0.10 ml (HBSS) using a 1 cc syringe fitted with 25 g needle.All animals were manipulated in a laminar flow hood and all cages,bedding, food and water were autoclaved. Tumor cells were passaged byexcising tumors and passing these through a 40 mesh screen; cells werewashed twice with 1× HBSS (50 ml) by centrifugation (1300 RPM),resuspended in I× HBSS to 10×10⁶ cells/ml, and frozen at -70° C. untilused.

For the experimental conditions, cells from several frozen lots werethawed, pelleted by centrifugation (1300 RPM) and washed twice with 1×HBSS. Cells were then resuspended to approximately 2.0×10⁶ cells/ml.Approximately 9 to 12 mice were injected with 0.10 ml of the cellsuspension (s.c.) using a 1 cc syringe fitted with a 25 g needle;injections were made on the animal's left side, approximatelymid-region. Tumors developed in approximately two weeks. Tumors wereexcised and processed as described above. Study mice were injected asdescribed above with 1.67×10⁶ cells in 0.10 ml HBSS.

Based on preliminary dosing experiments, it was determined that 200 mgof C2B8 and 100μ Ci of Y2B8 would be utilized for the study. Ninetyfemale nu/nu mice (approximately 10 weeks old) were injected with thetumor cells. Approximately ten days later, 24 mice were assigned to fourstudy groups (six mice/group) while attempting to maintain a comparabletumor size distribution in each group (average tumor size, expressed asa product of length×width of the tumor, was approximately 80 mm²). Thefollowing groups were treated as indicated via tail-vain injectionsusing a 100 μl Hamilton syringe fitted with a 25 g needle:

A. Normal Saline

B. Y2B8 (100μ Ci)

C. C2B8 (200 μg); and

D. Y2B8 (100μ Ci)+C2B8 (200 μg)

Groups tested with C2B8 were given a second C2B8 injection (200μg/mouse) seven days after the initial injection. Tumor measurementswere made every two or three days using a caliper.

Preparation of treatment materials were in accordance with the followingprotocols:

A Preparation of Y2B8

Yttrium- 90! chloride (6 mCi) was transformed to a polypropylene tubeand adjusted to pH 4.1-4.4 using metal free 2M sodium acetate.2B8-MX-DTPA (0.3 mg in normal saline; see above for preparation of2B8-MX-DTPA) was added and gently mixed by vortexing. After 15 min.incubation, the reaction was quenched by adding 0.05×volume 20 mM EDTAand 0.05×volume 2M sodium acetate. Radioactivity concentration wasdetermined by diluting 5.0 μl of the reaction mixture in 2.5 ml 1× PBScontaining 75 mg/ml HSA And 1 mM DTPA ("formulation buffer"); countingwas accomplished by adding 10.0 μl to 20 ml of Ecolume™ scintillationcocktail. The remainder of the reactive mixture was added to 3.0 mlformulation buffer, sterile filtered and stored at 2°-8° C. until used.Specific activity (14 mCi/mg at time of injection) was calculated usingthe radioactivity concentration and the calculated protein concentrationbased upon the amount of antibody added to the reaction mixture.Protein-associated radioactivity was determined using instant thin-layerchromatography. Radioincorporation was 95%. Y2B8 was diluted informulation buffer immediately before use and sterile-filtered (finalradioactivity concentration was 1.0 mCi/ml).

B. Preparation of C2B8

C2B8 was prepared as described above. C2B8 was provided as a sterilereagent in normal saline at 5.01 mg/ml. Prior to injection, the C2B8 wasdiluted in normal saline to 2.0 mg/ml and sterile filtered.

C. Results

Following treatment, tumor size was expressed as a product of length andwidth, and measurements were taken on the days indicated in FIG. 11(Y2B8 vs. Saline); FIG. 12 (C2B8 vs. Saline); and FIG. 13 (Y2B8+C2B8 vs.Saline). Standard error was also determined.

As indicated in FIG. 13, the combination of Y2B8 and C2B8 exhibitedtumoricidal effects comparable to the effects evidenced by either Y2B8or C2B8.

V. ALTERNATIVE THERAPY STRATEGIES

Alternative therapeutic strategies recognized in view of the foregoingexamples are evident. One such strategy employs the use of a therapeuticdose of C2B8 followed within about one week with a combination of either2B8 and radioabeled 2B8 (eg, Y2B8); or 2B8, C2B8 and, eg Y2B8; or C2B8and, eg Y2B8. An additional strategy is utilization of radiolabeledC2B8--such a strategy allows for utilization of the benefits of theimmunologically active portion of C2B8 plus those benefits associatedwith a radiolabel. Preferred radiolabels include yttrium-90 given thelarger circulating half-life of C2B8 versus the murine antibody 2B8.Because of the ability of C2B8 to deplete B-cells, and the benefits tobe derived from the use of a radiolabel, a preferred alternativestrategy is to treat the patient with C2B8 (either with a single dose ormultiple doses) such that most, if not all, peripheral B cells have beendepleted. This would then be followed with the use of radiolabeled 2B8;because of the depletion of peripheral B cells, the radiolabeled 2B8stands an increased chance of targeting tumor cells. Iodine 131! labeled2B8 is preferably utilized, given the types of results reported in theliterature with this label (see Kaminski). An alternative preferenceinvolves the use of a radiolabeled 2B8 (or C2B8) first in an effort toincrease the permeability of a tumor, followed by single or multipletreatments with C2B8; the intent of this strategy is to increase thechances of the C2B8 in getting both outside and inside the tumor mass. Afurther strategy involved the use of chemotherapeutic agenst incombination with C2B8. These strategies include so-called "staggered"treatments, ie, treatment with chemotherapeutic agent, followed bytreatment with C2B8, followed by a repetition of this protocol.Alternatively, initial treatment with a single or multiple doses ofC2B8, thereafter followed with chemotherapeutic treatement, is viable.Preferred chemotherapeutic agents include, but are not limited to:cyclophlsphamide; doxorubicin; vincristine; and prednisone, SeeArmitage, J. O. et al., Cancer 50:1695 (1982), incorporated herein byreference.

The foregoing alternative therapy strategies are not intended to belimiting, but rather are presented as being representative.

VI. DEPOSIT INFORMATION

Anti-CD20 in TCAE 8 (transformed in E. coli for purposes of deposit) wasdeposited with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md., 20852, under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure ("Budapest Treaty").The microorganism was deposited with the ATCC on Nov. 4, 1992 and testedby the ATCC on Nov. 9, 1992, and determined to be viable on that date.The ATCC has assigned this microorganism for the following ATCC depositnumber: ATCC 69119 (anti-CD20 in TCAE 8). Hybridoma 2B8 was depositedwith the ATCC on Jun. 22, 1993 under the provisions of the BudapestTreaty. The viability of the culture was determined on Jun. 25, 1993 andthe ATCC has assigned this hybridoma the following ATCC deposit number:HB 11388.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGGAGCTTGGATCGATCCTCTATGGTT27                                                 (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8540 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GACGTCGCGGCCGCTCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAG60                AGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGGC120               GGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACT180               ATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGG240               GACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCT300               GGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCCT360               AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC420               GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG480               ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA540               TGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA600               AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC660               ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC720               ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGA780               TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGG840               GACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA900               CGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGC960               CATCACAGATCTCTCACCATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGG1020              CTCCCAGGTGCACGATGTGATGGTACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCA1080              TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG1140              TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC1200              CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC1260              AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC1320              TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG1380              TGTTGAATTCAGATCCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACAACATGC1440              GGCCGTGATATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT1500              GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC1560              TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT1620              GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT1680              GCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCTATGCCAAGTACGCCCCCTATTG1740              ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT1800              TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTT1860              GGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACC1920              CCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC1980              GTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATA2040              TAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACTGAACACAGACCCGTCGAC2100              ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCGCTAGCACC2160              AAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG2220              GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA2280              GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC2340              TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC2400              AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTTGT2460              GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC2520              TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA2580              TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC2640              GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC2700              CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGACTACAAG2760              TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA2820              GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAGG2880              AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG2940              TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC3000              GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG3060              AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC3120              CTCTCCCTGTCTCCGGGTAAATGAGGATCCGTTAACGGTTACCAACTACCTAGACTGGAT3180              TCGTGACAACATGCGGCCGTGATATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTT3240              GCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTC3300              CCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATT3360              CTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCA3420              GGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCGCTGGATCT3480              CCCGATCCCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATT3540              AATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAG3600              AGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGA3660              GACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAA3720              GCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGG3780              GCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTT3840              CTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATTTCGCGC3900              CAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCCATCATG3960              GTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAGAACGGA4020              GACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACCACAACC4080              TCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTCTCCATT4140              CCTGAGAACAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAACTCAAA4200              GAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATT4260              GAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGTTCTGTT4320              TACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATCATGCAG4380              GAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTTCTCCCA4440              GAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAGTTTGAA4500              GTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTCCTAAAG4560              TCATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCAGCCTCGACTGTGCC4620              TTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG4680              TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG4740              GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGA4800              CAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGAGCTAC4860              TAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATTAATTTTAAC4920              ACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAGAGACAGTGT4980              TCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGAGACTCCTAA5040              GCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAAGCCTGATTC5100              CGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGGGCAGGAGCC5160              AGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTTCTGACATAG5220              TTGTGTTGGGAGCTTGGATCGATCCTCTATGGTTGAACAAGATGGATTGCACGCAGGTTC5280              TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTG5340              CTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGAC5400              CGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGC5460              CACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTG5520              GCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGA5580              GAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTG5640              CCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGG5700              TCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTT5760              CGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGC5820              CTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG5880              GCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGA5940              GCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTTCCCGATT6000              CGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTT6060              CGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGC6120              CTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCA6180              GCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAA6240              TGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCA6300              TTCTAGTTGTGGTTTGTCCAAACTCATCAATCTATCTTATCATGTCTGGATCGCGGCCGC6360              GATCCCGTCGAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT6420              CCGCTCACAATTCCACACAACATACGAGCCGGAGCATAAAGTGTAAAGCCTGGGGTGCCT6480              AATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAA6540              ACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA6600              TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC6660              GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG6720              CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT6780              TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA6840              GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT6900              CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC6960              CTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG7020              TCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT7080              TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG7140              CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA7200              AGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA7260              AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG7320              GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAG7380              AAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG7440              GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT7500              GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCT7560              TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC7620              TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA7680              TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG7740              GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATT7800              GTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCA7860              TTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTT7920              CCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT7980              TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGG8040              CAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTG8100              AGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG8160              CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAA8220              AACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGT8280              AACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGT8340              GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT8400              GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCA8460              TGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACAT8520              TTCCCCGAAAAGTGCCACCT8540                                                      (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9209 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GACGTCGCGGCCGCTCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAG60                AGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGGC120               GGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACT180               ATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGG240               GACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCT300               GGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCCT360               AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC420               GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG480               ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA540               TGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCA600               AGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC660               ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC720               ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATACCGGTTTGACTCACGCGGA780               TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGG840               GACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA900               CGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTGAACCGTCAGATCGCCTGGAGACGC960               CATCACAGATCTCTCACTATGGATTTTCAGGTGCAGATTATCAGCTTCCTGCTAATCAGT1020              GCTTCAGTCATAATGTCCAGAGGACAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCT1080              GCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTACATC1140              CACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGCCACATCCAAC1200              CTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTACTCTCTC1260              ACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGT1320              AACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCA1380              TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTG1440              TGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC1500              CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC1560              AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC1620              TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG1680              TGTTGAATTCAGATCCGTTAACGGTTACCAACTACCTAGACTGGATTCGTGACAACATGC1740              GGCCGTGATATCTACGTATGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT1800              GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC1860              TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT1920              GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT1980              GCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCTATGCCAAGTACGCCCCCTATTG2040              ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT2100              TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTT2160              GGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACC2220              CCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTC2280              GTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATA2340              TAAGCAGAGCTGGGTACGTCCTCACATTCAGTGATCAGCACTGAACACAGACCCGTCGAC2400              ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCTGTCCCAG2460              GTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCC2520              TGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAACAGACACCT2580              GGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAAT2640              CAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATG2700              CAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTAC2760              TACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCA2820              GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGG2880              GGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCG2940              TGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCA3000              GGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACC3060              TACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCC3120              AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA3180              CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT3240              GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGG3300              TACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC3360              AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG3420              GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC3480              AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAG3540              CTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC3600              GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG3660              CTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG3720              CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACG3780              CAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGGATCCGTTAACGGTTACCAACTACCT3840              AGACTGGATTCGTGACAACATGCGGCCGTGATATCTACGTATGATCAGCCTCGACTGTGC3900              CTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG3960              GTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTA4020              GGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG4080              ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGC4140              GCTGGATCTCCCGATCCCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAA4200              AGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGG4260              ATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACC4320              CAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTC4380              ATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGG4440              ATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCA4500              CATTTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCG4560              ATTTCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCT4620              GCCATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGC4680              AAGAACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATG4740              ACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGG4800              TTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGA4860              GAACTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTA4920              AGACTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGC4980              AGTTCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGG5040              ATCATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAA5100              CTTCTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTAT5160              AAGTTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCC5220              CTCCTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCAGCCTC5280              GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC5340              CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG5400              TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGA5460              TTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGC5520              TCGAGCTACTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGGAAAATT5580              AATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATGCTTTAG5640              AGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAGAGCTGA5700              GACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATCACCGAA5760              GCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATAGAGAGG5820              GCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACATTTGCTT5880              CTGACATAGTTGTGTTGGGAGCTTGGATCGATCCTCTATGGTTGAACAAGATGGATTGCA5940              CGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGAC6000              AATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTT6060              TGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATC6120              GTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGG6180              AAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGC6240              TCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCC6300              GGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGAT6360              GGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGC6420              CGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCA6480              TGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGA6540              CTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATAT6600              TGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGC6660              TCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACT6720              CTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCC6780              ACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATG6840              ATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCA6900              GCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTT6960              TCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATCTATCTTATCATGTCTGGATC7020              GCGGCCGCGATCCCGTCGAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAA7080              ATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCT7140              GGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCC7200              AGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCG7260              GTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC7320              GGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG7380              GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA7440              AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC7500              GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCC7560              CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCG7620              CCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTT7680              CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC7740              GCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC7800              CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAG7860              AGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG7920              CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA7980              CCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAG8040              GATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT8100              CACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA8160              ATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTT8220              ACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAG8280              TTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA8340              GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC8400              AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT8460              CTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACG8520              TTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCA8580              GCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG8640              TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCA8700              TGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTG8760              TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT8820              CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCA8880              TCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA8940              GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCG9000              TTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACAC9060              GGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT9120              ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC9180              CGCGCACATTTCCCCGAAAAGTGCCACCT9209                                             (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 47 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATCACAGATCTCTCACCATGGATTTTCAGGTGCAGATTATCAGCTTC47                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TGCAGCATCCGTACGTTTGATTTCCAGCTT30                                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 384 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..384                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 67..384                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATGGATTTTCAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGCTTCA48                            MetAspPheGlnValGlnIleIleSerPheLeuLeuIleSerAlaSer                              22-20-15-10                                                                   GTCATAATGTCCAGAGGGCAAATTGTTCTCTCCCAGTCTCCAGCAATC96                            ValIleMetSerArgGlyGlnIleValLeuSerGlnSerProAlaIle                              5-11510                                                                       CTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGC144                           LeuSerAlaSerProGlyGluLysValThrMetThrCysArgAlaSer                              152025                                                                        TCAAGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCCTCC192                           SerSerValSerTyrIleHisTrpPheGlnGlnLysProGlySerSer                              303540                                                                        CCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCCCT240                           ProLysProTrpIleTyrAlaThrSerAsnLeuAlaSerGlyValPro                              455055                                                                        GTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTACTCTCTCACAATC288                           ValArgPheSerGlySerGlySerGlyThrSerTyrSerLeuThrIle                              606570                                                                        AGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGG336                           SerArgValGluAlaGluAspAlaAlaThrTyrTyrCysGlnGlnTrp                              75808590                                                                      ACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAA384                           ThrSerAsnProProThrPheGlyGlyGlyThrLysLeuGluIleLys                              95100105                                                                      (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCGGCTCCCACGCGTGTCCTGTCCCAG27                                                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /note= "Nucleotide 3 is N wherein N                    is G or C."                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note= "Nucleotide 18 is N wherein                     N is A or C."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 19                                                              (D) OTHER INFORMATION: /note= "Nucleotide 19 is N wherein                     N is A or G."                                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 25                                                              (D) OTHER INFORMATION: /note= "Nucleotide 25 is N wherein                     N is G or A."                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGNTGTTGTGCTAGCTGNNGAGACNGTGA29                                               (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..420                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 58..420                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGT48                            MetGlyTrpSerLeuIleLeuLeuPheLeuValAlaValAlaThrArg                              19- 15-10-5                                                                   GTCCTGTCCCAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAG96                            ValLeuSerGlnValGlnLeuGlnGlnProGlyAlaGluLeuValLys                              11510                                                                         CCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTT144                           ProGlyAlaSerValLysMetSerCysLysAlaSerGlyTyrThrPhe                              152025                                                                        ACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTG192                           ThrSerTyrAsnMetHisTrpValLysGlnThrProGlyArgGlyLeu                              30354045                                                                      GAATGGATTGGAGCTATTTATCCCGGAAATGGTGATACTTCCTACAAT240                           GluTrpIleGlyAlaIleTyrProGlyAsnGlyAspThrSerTyrAsn                              505560                                                                        CAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGC288                           GlnLysPheLysGlyLysAlaThrLeuThrAlaAspLysSerSerSer                              657075                                                                        ACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTC336                           ThrAlaTyrMetGlnLeuSerSerLeuThrSerGluAspSerAlaVal                              808590                                                                        TATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAAT384                           TyrTyrCysAlaArgSerThrTyrTyrGlyGlyAspTrpTyrPheAsn                              95100105                                                                      GTCTGGGGCGCAGGGACCACGGTCACCGTCTCTGCA420                                       ValTrpGlyAlaGlyThrThrValThrValSerAla                                          110115120                                                                     __________________________________________________________________________

What is claimed is:
 1. A method of treating B-cell lymphoma whichincludes the following steps: (i) administering, at a firstadministration period, a radiolabeled anti-CD20 antibody, wherein saidantibody is selected from the group consisting of the chimeric anti-CD20antibody produced by transfectoma TCAE 8, which has been accordedAmerican Type Culture Collection Deposit No. ATCC 69119, and the murineanti-CD20 antibody secreted by a hybridoma identified by American TypeCulture Collection Deposit No. HB 11388; and (ii) subsequentlyadministering after step (i) at least one dosage of a chimeric anti-CD20antibody produced by transfectoma TCAE 8, which has been accordedAmerican Type Culture Collection Deposit No. ATCC
 69119. 2. The methodof claim 1, wherein multiple dosages of said chimeric anti-CD20antibodies are administered after said initial administration (i) ofsaid radiolabeled anti-CD20 antibody.
 3. The method of claim 1, whereinsaid radiolabeled antibody administered in step (i) is a radiolabeledmurine anti-CD20 antibody, and said murine anti-CD20 antibody issecreted by the hybridoma identified by American Type Culture CollectionDeposit No. HB
 11388. 4. The method of claim 1, wherein saidradiolabeled antibody administered in step (i) is a radiolabeledchimeric anti-CD20 antibody, wherein said chimeric anti-CD20 antibody isproduced by transfectoma TCAE 8, which has been accorded American TypeCulture Collection Deposit No. ATCC
 69119. 5. The method of claim 1,which further includes harvesting of peripheral stem cells (PSC's) orbone marrow (BM) prior to treatment.
 6. The method of claim 5, whereinsaid harvested PSC's or BM is reinfused after treatment.
 7. The methodof claim 1, wherein the radiolabel contained in said radiolabeledantibody administered in step (i) is selected from the group consistingof iodine (131), indium (131), indium (111), and yttrium (90).